Preparation and therapeutic applications of (2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]OCT-3-yl)-3,5-difluorobenzamide

ABSTRACT

The present invention relates to compounds that bind to and modulate the activity of neuronal nicotinic acetylcholine receptors, to processes for preparing these compounds, to pharmaceutical compositions containing these compounds, and to methods of using these compounds for treating a wide variety of conditions and disorders, including those associated with dysfunction of the central nervous system (CNS).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a §371 Application of PCT Application No.PCT/2010/021926, filed Jan. 25, 2010, which claims priority to U.S.Provisional Application Ser. No. 61/147,260, filed Jan. 26, 2009, eachof which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compounds that bind to and modulate theactivity of neuronal nicotinic acetylcholine receptors, to processes forpreparing these compounds, to pharmaceutical compositions containingthese compounds, and to methods of using these compounds for treating awide variety of conditions and disorders, including those associatedwith dysfunction of the central nervous system (CNS).

BACKGROUND OF THE INVENTION

The therapeutic potential of compounds that target neuronal nicotinicreceptors (NNRs), also known as nicotinic acetylcholine receptors(nAChRs), has been the subject of several reviews (see, for example,Breining et al., Ann. Rep. Med. Chem. 40: 3 (2005), Hogg and Bertrand,Curr. Drug Targets: CNS Neurol. Disord. 3: 123 (2004), Suto andZacharias, Expert Opin. Ther. Targets 8: 61 (2004), Dani et al., Bioorg.Med. Chem. Lett. 14: 1837 (2004), Bencherif and Schmitt, Curr. DrugTargets: CNS Neurol. Disord. 1: 349 (2002)). Among the kinds ofindications for which NNR ligands have been proposed as therapies arecognitive disorders, including Alzheimer's disease, attention deficitdisorder, and schizophrenia (Newhouse et al., Curr. Opin. Pharmacol. 4:36 (2004), Levin and Rezvani, Curr. Drug Targets: CNS Neurol. Disord. 1:423 (2002), Graham et al., Curr. Drug Targets: CNS Neurol. Disord. 1:387 (2002), Ripoll et al., Curr. Med. Res. Opin. 20(7): 1057 (2004), andMcEvoy and Allen, Curr. Drug Targets: CNS Neurol. Disord. 1: 433(2002)); pain and inflammation (Decker et al., Curr. Top. Med. Chem.4(3): 369 (2004), Vincler, Expert Opin. Invest. Drugs 14(10): 1191(2005), Jain, Curr. Opin. Inv. Drugs 5: 76 (2004), Miao et al.,Neuroscience 123: 777 (2004)); depression and anxiety (Shytle et al.,Mol. Psychiatry 7: 525 (2002), Damaj et al., Mol. Pharmacol. 66: 675(2004), Shytle et al., Depress. Anxiety 16: 89 (2002));neurodegeneration (O'Neill et al., Curr. Drug Targets: CNS Neurol.Disord. 1: 399 (2002), Takata et al., J. Pharmacol. Exp. Ther. 306: 772(2003), Marrero et al., J. Pharmacol. Exp. Ther. 309: 16 (2004));Parkinson's disease (Jonnala and Buccafusco, J. Neurosci. Res. 66: 565(2001)); addiction (Dwoskin and Crooks, Biochem. Pharmacol. 63: 89(2002), Coe et al., Bioorg. Med. Chem. Lett. 15(22): 4889 (2005));obesity (Li et al., Curr. Top. Med. Chem. 3: 899 (2003)); and Tourette'ssyndrome (Sacco et al., J. Psychopharmacol. 18(4): 457 (2004), Young etal., Clin. Ther. 23(4): 532 (2001)).

There exists a heterogeneous distribution of nAChR subtypes in both thecentral and peripheral nervous systems. For instance, the nAChR subtypeswhich are predominant in vertebrate brain are α4β2, α7, and α3β2,whereas those which predominate at the autonomic ganglia are α3β4 andthose of neuromuscular junction are α1β1δγ and α1β1δε (see Dwoskin etal., Exp. Opin. Ther. Patents 10: 1561 (2000) and Holliday et al. J.Med. Chem. 40(26), 4169 (1997)).

A limitation of some nicotinic compounds is that they are associatedwith various undesirable side effects due to non-specific binding tomultiple nAChR subtypes. For example, binding to and stimulation ofmuscle and ganglionic nAChR subtypes can lead to side effects which canlimit the utility of a particular nicotinic binding compound as atherapeutic agent.

The compounds of the present invention exhibit a high degree of specificbinding to the α7 nAChR subtype and low affinity for the α4β2 subtype aswell as ganglionic and muscle nAChR subtypes. Thus, these compounds canserve as therapeutic modulators of α7 nAChRs in patients in need of suchtreatment, without producing side effects caused by non-specific nAChRsubtype binding.

SUMMARY OF THE INVENTION

The present invention includes(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(Formula I) or a pharmaceutically acceptable salt thereof.

The compound of the present invention binds with high affinity to NNRsof the α7 subtype and exhibit selectivity for this subtype over the α4β2NNR subtype, as well as over ganglion and muscle subtypes.

The present invention includes pharmaceutical compositions comprisingthe compound of the present invention or a pharmaceutically acceptablesalt thereof. The pharmaceutical compositions of the present inventioncan be used for treating or preventing a wide variety of conditions ordisorders, including those disorders characterized by dysfunction ofnicotinic cholinergic neurotransmission or the degeneration of thenicotinic cholinergic neurons.

The present invention includes a method for treating or preventingdisorders and dysfunctions, such as CNS disorders and dysfunctions,inflammation, inflammatory response associated with bacterial and/orviral infection, pain, metabolic syndrome, autoimmune disorders, orother disorders described in further detail herein. The presentinvention includes a method for modulating neovascularization. Themethods involve administering to a subject a therapeutically effectiveamount of a compound of the present invention, including a salt thereof,or a pharmaceutical composition that includes such compounds.Additionally the present invention includes compounds that have utilityas diagnostic agents and in receptor binding studies as describedherein.

The foregoing and other aspects of the present invention are explainedin further detail in the detailed description and examples set forthbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts novel object recognition (NOR) vs. dose for(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideor pharmaceutically acceptable salt thereof. A statistically significanteffect was observed for doses as low as 0.1 mg/kg.

FIG. 2 depicts the data used for the determination of the minimumeffective dose for novel object recognition (NOR) upon administration of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideor pharmaceutically acceptable salt thereof. A statistically significanteffect was observed for doses as low as 0.03 mg/kg.

FIG. 3 depicts novel object recognition (NOR) vs. time following the 3rdadministration of 0.1 mg/kg(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideor a pharmaceutically acceptable salt thereof. A statisticallysignificant effect was observed for doses out to 6 h after dosing.

FIG. 4 depicts novel object recognition (NOR) vs. time following the 3rdadministration of 0.3 mg/kg(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideor a pharmaceutically acceptable salt thereof. A statisticallysignificant effect was observed for doses out to 18 h after dosing.

FIG. 5 depicts a dose response for each of Compound A and Compound Bwith α7 nicotinic receptors.

FIG. 6 depicts the electrophysiological response to co-application ofeach of Compound A and Compound B with acetylcholine (Ach).

FIGS. 7A, 7B, and 7C depict electrophysiological response forinteraction of Compound A with Ach, regarding activation of thenicotinic α7 receptor.

FIGS. 8A, 8B, and 8C depict electrophysiological response forinteraction of Compound B with Ach, regarding activation of thenicotinic α7 receptor.

FIG. 9 is an x-ray diffraction pattern for Compound A mono-hydrochloridesalt.

FIG. 10 is a crystal structure for Compound A mono-hydrochloride salt.

FIG. 11 is an x-ray diffraction pattern for Compound A hemi-galactaratesalt.

FIG. 12 illustrates an overlay of six (6) different x-ray diffractionpatterns for salts from the salt screen for Compound A.

FIG. 13 illustrates the results of assessment of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidein CFA-induced thermal hyperalgesia. Test substance, morphine, andvehicle were each administered subcutaneously to groups of 8 SD rats 24hours after CFA injection. The thermal hyperalgesia was performed priorto CFA injection (pre-CFA). before treatment, and 1 hour after SCinjection. One-way ANOVA followed by the Dunnett's test was applied bcompare between the treatment groups and the vehicle controlled group.Differences are considered significant at the *P<0.05 level.

FIG. 14 illustrates the results of Von Frey assessment indicating that(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideis effective in reducing diabetic neuropathy pain at doses of 1 mg/kgand 10 mg/kg compared to the Vehicle treated group.

FIG. 15 illustrates comparison weight gain as significantly lower in the(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide-treatedobese (“db-Test Article”) mice. Notably, animals that wereco-administered MLA with(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidefailed to show the reduced weight gain exhibited by the obese ratsadministered(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidealone.

FIG. 16 illustrates average food consumption was significantly lower inthe(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide-treatedobese mice (“db-Test Article”) than in the obese controls. The foodconsumption of the lean mice was unaffected by(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(“Db-Test Article”). Animals that were co-administered MLA with(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidefailed to show the reduced daily average food consumption exhibited bythe obese rats administered(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidealone.

FIG. 17 illustrates that(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidesignificantly inhibited fasting plasma glucose levels in obese mice(“db-Test Article”). However, this effect was not reversed byco-administration with MLA.

FIG. 18 illustrates that(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidesignificantly inhibited glycosylated HbA1c levels in obese mice(“db-Test Article”). The reduction in glycosylated HbA1c by(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidewas attenuated by co-administration of MLA.

FIG. 19 illustrates that(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidesignificantly reduced the pro-inflammatory cytokine TNF alpha in obesemice (“db-Test Article”). These effects were inhibited byco-administration of the alpha7 antagonist MLA.

FIG. 20 illustrates that(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideresulted in significantly lower triglyceride levels in obese mice(“db-Test Article”) compared with vehicle-treated controls (“db”). Thereduction in triglycerides by(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidewas not attenuated by co-administration of MLA.

FIG. 21 illustrates the effect of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideon % changes in Penh response to methacholine challenge inovalbumin-sensitized mice.(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideand vehicle were administered subcutaneously bid or givenintratracheally qd for 6 consecutive days from day 21 to day 25 at 30min before OVA challenge and the last dosing was administrated at 30 minbefore MCh provocation on day 26. The Penh values were determined.One-way ANOVA followed by Dunnett's test was applied for comparisonbetween the OVA immunized vehicle and other treatment groups. *P<0.05vs. OVA-vehicle control.

FIG. 22 illustrates the effect of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideon white blood cell counts and differential cell counts in ovalbuminsensitized mice.(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideand vehicle were administered subcutaneously bid or were givenintratracheally qd for 6 consecutive days from day 21 to day 25 at 30minutes before OVA challenge and the last dosing was administrated at 30minutes before bronchoalveolar lavage fluid harvest on day 26. The totalwhite blood cell count and differential cell counts were determined.One-way ANOVA followed by Dunnett's test was applied for comparisonbetween the OVA immunized vehicle and other treatment groups. *P<0.05vs. OVA-vehicle control.

FIG. 23 illustrates the effect of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideon % white blood cell count and differential cell counts in ovalbuminsensitized mice.(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideand vehicle were administered subcutaneously bid or were givenintratracheally qd for 6 consecutive days from day 21 to day 25 at 30minutes before OVA challenge and the last dosing was administrated at 30minutes before bronchoalveolar lavage fluid harvest on day 26. The totalwhite blood cell count and differential cell counts were determined.One-way ANOVA followed by Dunnett's test was applied for comparisonbetween the OVA immunized vehicle and other treatment groups. *P<0.05vs. OVA-vehicle control.

DETAILED DESCRIPTION

Definitions

The following definitions are meant to clarify, but not limit, the termsdefined. If a particular term used herein is not specifically defined,such term should not be considered indefinite. Rather, terms are usedwithin their accepted meanings.

As used herein, the term “compound(s)” may be used to mean the free baseform, or alternatively, a salt form of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide,depending on the context, which will be readily apparent. Those skilledin the art will be able to distinguish the difference.

For ease of reference,(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(Formula I) or a pharmaceutically acceptable salt thereof is alsoreferred to as Compound A. Additionally, a structural analog is usedherein for comparative purposes.(2S,3R)-N-2-((3-Pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-4-fluorobenzamideor a pharmaceutically acceptable salt thereof is referred to as CompoundB. Compound B is a single isomer of a racemic mixture as published in WO04/76449, herein incorporated by reference.

As used herein, the term “pharmaceutically acceptable” refers tocarrier(s), diluent(s), excipient(s) or salt forms of the compound ofthe present invention that are compatible with the other ingredients ofthe formulation and not deleterious to the recipient of thepharmaceutical composition.

As used herein, the term “pharmaceutical composition” refers to acompound of the present invention optionally admixed with one or morepharmaceutically acceptable carriers, diluents, or exipients.Pharmaceutical compositions preferably exhibit a degree of stability toenvironmental conditions so as to make them suitable for manufacturingand commercialization purposes.

As used herein, the terms “effective amount”, “therapeutically effectiveamount”, “therapeutic amount,” or “effective dose” refer to an amount ofthe compound of the present invention sufficient to elicit the desiredpharmacological or therapeutic effects, thus resulting in effectiveprevention or treatment of a disorder. Prevention of the disorder may bemanifested by delaying or preventing the progression of the disorder, aswell as the onset of the symptoms associated with the disorder.Treatment of the disorder may be manifested by a decrease or eliminationof symptoms, inhibition or reversal of the progression of the disorder,as well as any other contribution to the well being of the patient.

As will be discussed in more detail below and with reference to FIGS. 12, 3, and 4, a statistically significant effect is observed for doses ofthe compound of Formula I, or a pharmaceutically acceptable saltthereof, as low as 0.03 μM/kg, including effects observed out to 18hours after dosing. The effective dose can vary, depending upon factorssuch as the condition of the patient, the severity of the symptoms ofthe disorder, and the manner in which the pharmaceutical composition isadministered. Thus, as used herein, the effective dose may be less than100 mg, preferably less than 50 mg, more preferably less than 10 mg, andmost preferably less than 1 mg. These effective doses typicallyrepresent the amount administered as a single dose, or as one or moredoses administered over a 24 hours period.

Compounds

One aspect of the present invention includes a compound(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(Formula I) or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound is substantially free of one or more of(2R,3S)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide,(2R,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide,and(2S,3S)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide.

In one embodiment, the is an acid addition salt, wherein the acid isselected from hydrochloric acid, methanesulfonic acid, maleic acid,phosphoric acid, 1-hydroxy-2-naphthoic acid, malonic acid, L-tartaricacid, fumaric acid, citric acid, L-malic acid, R-mandelic acid,S-mandelic acid, succinic acid, 4-acetamidobenzoic acid, adipic acid,galactaric acid, di-p-toluoyl-D-tartaric acid, oxalic acid, D-glucuronicacid, 4-hydroxybenzoic acid, 4-methoxybenzoic acid,(1S)-(+)-10-camphorsulfonic acid, (1R,3S)-(+)-camphoric acid, andp-toluenesulfonic acid, or a hydrate or solvate thereof. In a furtherembodiment, the molar ratio of acid to(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideis 1:2 or 1:1.

Another aspect of the present invention includes a compound selectedfrom:

(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidemono-hydrochloride or a hydrate or solvate thereof;

(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidemono-phosphate or a hydrate or solvate thereof;

(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidemono-4-hydroxybenzoate or a hydrate or solvate thereof; and

(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidehemi-4-hydroxybenzoate or a hydrate or solvate thereof.

Another aspect of the present invention includes a compound,(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideor a pharmaceutically acceptable salt thereof containing less than 25%,preferably containing less than 15%, preferably containing less than 5%,preferably containing less than 2%, preferably containing containingless than 1% of (2R,3R)-, (2S,3S)-, or(2R,3S)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide,either individually or in combination, by weight.

Another aspect of the present invention includes a compound(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(Formula I) or a pharmaceutically acceptable salt thereof which issubstantially crystalline. Another aspect includes a polymorphic form ofa compound(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidehydrochloride characterized by an x-ray diffraction pattern comprisingone or more peaks within ±0.5 degrees 29θ of the following peaks:

2θ 8.4 8.8 11.9 13.2 15.2 16.0 17.6 18.4 18.9 19.9 20.1 21.3 23.1 25.426.2

Another aspect of the present invention is a polymorphic form of acompound(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidehydrochloride characterized by an x-ray powder diffraction pattern thatsubstantially corresponds to FIG. 9.

Another aspect of the present invention includes use of a compound ofthe present invention, in the manufacture of a medicament for thetreatment or prevention of an α7-mediated disease or dysfunction.Another aspect of the present invention includes a method for treatingor preventing an α7-mediated disease or dysfunction, comprisingadministering a therapeutically effective amount of a compound of thepresent invention. Another aspect of the present invention includes acompound of the present invention for use in treating or preventing anα7-mediated disease or dysfunction. In one embodiment, the disease ordysfunction is selected from the group consisting of:

i) pain, including one or more of acute, neurologic, inflammatory,neuropathic, chronic pain, severe chronic pain, post-operative pain,pain associated with cancer, angina, renal or biliary colic,menstruation, migraine, gout, arthritis, rheumatoid disease,teno-synovitis, vasculitis, trigeminal or herpetic, neuralgia, diabeticneuropathy pain, causalgia, low back pain, deafferentation syndromes,and brachial plexus avulsion;

ii) metabolic syndrome, weight gain, type I diabetes mellitus, type IIdiabetes mellitus, or diabetic neuropahy;

iii) inflammation, including one or more of psoriasis, asthma,atherosclerosis, idiopathic pulmonary fibrosis, chronic and acuteinflammation, psoriasis, endotoxemia, gout, acute pseudogout, acutegouty arthritis, arthritis, rheumatoid arthritis, osteoarthritis,allograft rejection, chronic transplant rejection, asthma,atherosderosis, mononuclear-phagocyte dependent lung injury, atopicdermatitis, chronic obstructive pulmonary disease, adult respiratorydistress syndrome, acute chest syndrome in sickle cell disease,inflammatory bowel disease, Crohn's disease, ulcerative colitis, acutecholangitis, aphteous stomatitis, pouchitis, glomerulonephritis, lupusnephritis, thrombosis, and graft vs. host reaction; and

iv) cognition, including one or more of age-associated memoryimpairment, mild cognitive impairment, pre-senile dementia, early onsetAlzheimer's disease, senile dementia, dementia of the Alzheimer's type,mild to moderate dementia of the Alzheimer's type, Lewy body dementia,vascular dementia, Alzheimer's disease, stroke, AIDS dementia complex,attention deficit disorder, attention deficit hyperactivity disorder,dyslexia, schizophrenia, schizophreniform disorder, schizoaffectivedisorder, cognitive deficits in schizophrenia, and cognitive dysfunctionin schizophrenia.

Another aspect of the present invention includes a pharmaceuticalcomposition comprising a compound of the present invention and one ormore pharmaceutically acceptable carrier.

Another aspect of the present invention includes a method of enhancingacetylcholine-induced current comprising administering an effectiveamount of a compound of the present invention.

Another embodiment of the present invention includes(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideor a pharmaceutically acceptable salt thereof with reference to any oneof the Examples.

Another embodiment of the present invention includes(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideor a pharmaceutically acceptable salt thereof for use as an activetherapeutic substance.

Another embodiment of the present invention includes a method ofmodulating NNR in a subject in need thereof through the administrationof(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideor a pharmaceutically acceptable salt thereof.

The scope of the present invention includes combinations of aspects andembodiments.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructure except for the replacement of a hydrogen atom by deuterium ortritium, or the replacement of a carbon atom by ¹³C or ¹⁴C, or thereplacement of a nitrogen atom by ¹⁵N, or the replacement of an oxygenatom with ¹⁷O or¹⁸O are within the scope of the invention. Suchisotopically labeled compounds are useful as research or diagnostictools.

The present invention includes a salt or solvate of the(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide,including combinations thereof, such as a solvate of a salt. Thecompounds of the present invention may exist in solvated, for examplehydrated, as well as unsolvated forms, and the present inventionencompasses all such forms.

Typically, but not absolutely, the salts of the present invention arepharmaceutically acceptable salts. Salts encompassed within the term“pharmaceutically acceptable salts” refer to non-toxic salts of thecompounds of this invention.

Examples of suitable pharmaceutically acceptable salts include inorganicacid addition salts such as chloride, bromide, sulfate, phosphate, andnitrate; organic acid addition salts such as acetate, galactarate,propionate, succinate, lactate, glycolate, malate, tartrate, citrate,maleate, fumarate, methanesulfonate, p-toluenesulfonate, and ascorbate;salts with acidic amino acid such as aspartate and glutamate; alkalimetal salts such as sodium salt and potassium salt; alkaline earth metalsalts such as magnesium salt and calcium salt; ammonium salt; organicbasic salts such as trimethylamine salt, triethylamine salt, pyridinesalt, picoline salt, dicyclohexylamine salt, andN,N′-dibenzylethylenediamine salt; and salts with basic amino acid suchas lysine salt and arginine salt. The salts may be in some caseshydrates or ethanol solvates.

As noted herein, the present invention includes specific compounds,which are identified herein with particularity. The compounds of thisinvention may be made by a variety of methods, including well-knownstandard synthetic methods. Illustrative general synthetic methods areset out below and then specific compounds of the invention are preparedin the working Examples.

In all of the examples described below, protecting groups for sensitiveor reactive groups are employed where necessary in accordance withgeneral principles of synthetic chemistry. Protecting groups aremanipulated according to standard methods of organic synthesis (see, forexample, T. W. Green and P. G. M. Wuts, Protecting Groups in OrganicSynthesis, 3^(rd) Edition, John Wiley & Sons, New York (1999)). Thesegroups are removed at a convenient stage of the compound synthesis usingmethods that are readily apparent to those skilled in the art. Theselection of processes as well as the reaction conditions and order oftheir execution shall be consistent with the preparation of compounds ofthe present invention.

The present invention also provides a method for the synthesis ofcompounds useful as intermediates in the preparation of compounds of thepresent invention along with methods for their preparation.

The compounds can be prepared according to the following methods usingreadily available starting materials and reagents. In these reactions,variants may be employed which are themselves known to those of ordinaryskill in this art, but are not mentioned in greater detail.

Salt Forms

One aspect of the present invention relates to novel salt forms of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide.

(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidein the free base form is a solid with limited water solubility. However,the free base will react with both inorganic and organic acids to makecertain acid addition salts that have physical properties that areadvantageous for the preparation of pharmaceutical compositions such ascrystallinity, water solubility, and stability toward chemicaldegradation. Typically, these salt forms are pharmaceutically acceptablesalts.

The present invention includes pharmaceutically acceptable salts of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide.Examples of suitable pharmaceutically acceptable salts include inorganicacid addition salts such as chloride, bromide, sulfate, phosphate, andnitrate; organic acid addition salts such as acetate, galactarate,propionate, succinate, lactate, glycolate, malate, tartrate, citrate,maleate, fumarate, methanesulfonate, p-toluenesulfonate, and ascorbate;salts with acidic amino acid such as aspartate and glutamate; alkalimetal salts such as sodium salt and potassium salt; alkaline earth metalsalts such as magnesium salt and calcium salt; ammonium salt; organicbasic salts such as trimethylamine salt, triethylamine salt, pyridinesalt, picoline salt, dicyclohexylamine salt, andN,N′-dibenzylethylenediamine salt; and salts with basic amino acid suchas lysine salt and arginine salt. The salts may be in some caseshydrates or solvates, such as ethanol solvates.

One aspect of the present invention includes acid addition salts of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidewherein the acid is selected from hydrochloric acid, methanesulfonicacid, maleic acid, phosphoric acid, 1-hydroxy-2-naphthoic acid, malonicacid, L-tartaric acid, fumaric acid, citric acid, L-malic acid,R-mandelic acid, S-mandelic acid, succinic acid, 4-acetamidobenzoicacid, adipic acid, galactaric acid, di-p-toluoyl-D-tartaric acid, oxalicacid, D-glucuronic acid, 4-hydroxybenzoic acid, 4-methoxybenzoic acid,(1S)-(+)-10-camphorsulfonic acid, (1R,3S)-(+)-camphoric acid, andp-toluenesulfonic acid. The present invention also includes hydrates andsolvates of these salt forms.

The stoichiometry of the salts comprising the present invention canvary. For example, it is typical that the molar ratio of acid to(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideis 1:2 or 1:1, but other ratios, such as 3:1, 1:3, 2:3, 3:2 and 2:1, arepossible.

In one embodiment of the present invention, the salt has a stoichiometryof acid to of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideof 1:2. In another embodiment, the salt has a stoichiometry of acid of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideof 1:1.

As herein noted, depending upon the manner by which the salts describedherein are formed, the salts can have crystal structures that occludesolvents that are present during salt formation. Thus, the salts canoccur as hydrates and other solvates of varying stoichiometry of solventrelative to(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide.

Another embodiment of the present invention includes(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideor a hydrate or solvate thereof.

Another embodiment of the present invention includes(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidemono-hydrochloride or a hydrate or solvate thereof.

Another embodiment of the present invention(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidemono-phosphate or a hydrate or solvate thereof.

Another embodiment of the present invention includes(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidemono-4-hydroxybenzoate or a hydrate or solvate thereof.

Another embodiment of the present invention includes(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidehemi-4-hydroxybenzoate or a hydrate or solvate thereof.

A further aspect of the present invention includes processes for thepreparation of the salts. The precise conditions under which the saltsare formed may be empirically determined. The salts may be obtained bycrystallization under controlled conditions.

One embodiment of the present invention includes a method for thepreparation of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideor a pharmaceutically acceptable salt thereof containing less than 25%,preferably less than 15%, more preferably less than 5%, even morepreferably less than 2%, and most preferably less than 1% of (2R,3R)-,(2S,3S)-, or (2R,3S)N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideby weight either individually or in combination.

The method for preparing the salt forms can vary. The preparation(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidesalt forms typically involves:

(i) mixing the free base, or a solution of the free base of suitablypure(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidein a suitable solvent, with any of the acids in pure form or as asolution of any of the acids in a suitable solvent, typically 0.5 to 1equivalents of the acid;

(ii) (a) cooing the resulting salt solution if necessary to causeprecipitation;

or

(ii) (b) adding a suitable anti-solvent to cause precipitation;

or

(ii) (c) evaporating the first solvent and adding and new solvent andrepeating either steps (ii) (a) or step (ii) (b);

and

(iii) filtering and collecting the salt.

The stoichiometry, solvent mix, solute concentration, and temperatureemployed can vary. Representative solvents that can be used to prepareor recrystallize the salt forms include, without limitation, ethanol,methanol, propanol, isopropyl alcohol, isopropyl acetate, acetone, ethylacetate, toluene, water, methyl ethyl ketone, methyl isobutyl ketone,tert-butyl methyl ether, tetrahydrofuran, dichloromethane, n-heptane,and acetonitrile.

Several of these salts demonstrate stability sufficient to establishtheir promise in the production of pharmaceutical preparations. Suchstability can be demonstrated in a variety of ways. Propensity to gainand release atmospheric moisture can be assessed by dynamic vaporsorption (DVS).

General Synthetic Methods

A synthesis of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideis achieved by O-(benzotriazol-1-yl)-N,N,N,1-tetramethyluroniumhexafluorophosphate (HBTU) mediated coupling of(2S,3R)-3-amino-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]octane(obtained as described in PCT/US08/71872, herein incorporated byreference with regard to such synthesis) and 3,5-difluorobenzoic acid asillustrated in Scheme 1.

The synthesis of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidecan be similarly achieved by the use of other agents to activate thecarboxylic acid. For example, the use of activating agents such asN,N′-dicyclohexylcarbodiimide (DCC),(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate(BOP), (benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBOP),O-(benzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (HBPyU),O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HBTU), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TBTU), and(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDCI) with1-hydroxybenzotriazole (HOBt), as well as those described in, forexample, Kiso and Yajima, Peptides, pp 39-91, Academic Press, San Diego,Calif. (1995), are well known to those skilled in the art.

Methods of Treatment

The compounds of the present invention have the ability to selectivelybind to and modulate the activity of α7 NNRs. Consequently, thesecompounds can be used for the prevention or treatment of variousconditions or disorders for which other types of nicotinic compoundshave been proposed or are shown to be useful as therapeutics, such asCNS disorders, inflammation, inflammatory response associated withbacterial and/or viral infection, pain, metabolic syndrome, autoimmunedisorders or other disorders described in further detail herein. Thesecompounds can be used for modulating neovascularization and asdiagnostic agents in receptor binding studies (in vitro and in vivo).Such therapeutic and other teachings are described, for example, inWilliams et al., Drug News Perspec. 7(4): 205 (1994), Arneric et al.,CNS Drug Rev. 1(1): 1-26 (1995), Arneric et al., Exp. Opin. Invest.Drugs 5(1): 79-100 (1996), Bencherif et al., J. Pharmacol. Exp. Ther.279: 1413 (1996), Lippiello et al., J. Pharmacol. Exp. Ther. 279: 1422(1996), Damaj et al., J. Pharmacol. Exp. Ther. 291: 390 (1999); Chiariet al., Anesthesiology 91: 1447 (1999), Lavand'homme and Eisenbach,Anesthesiology 91: 1455 (1999), Holladay et al., J. Med. Chem. 40(28):4169-94 (1997), Bannon et al., Science 279: 77 (1998), PCT WO 94/08992,PCT WO 96/31475, PCT WO 96/40682, and U.S. Pat. Nos. 5,583,140 toBencherif et al., 5,597,919 to Dull et al., 5,604,231 to Smith et al.and 5,852,041 to Cosford et al., and other references previously listedherein.

CNS Disorders

The compounds and their pharmaceutical compositions are useful in thetreatment or prevention of a variety of CNS disorders, includingneurodegenerative disorders, neuropsychiatric disorders, neurologicdisorders, and addictions. The compounds and their pharmaceuticalcompositions can be used to treat or prevent cognitive deficits anddysfunctions, age-related and otherwise; attentional disorders anddementias, including those due to infectious agents or metabolicdisturbances; to provide neuroprotection; to treat convulsions andmultiple cerebral infarcts; to treat mood disorders, compulsions andaddictive behaviors; to provide analgesia; to control inflammation, suchas mediated by cytokines and nuclear factor kappa B; to treatinflammatory disorders; to provide pain relief; and to treat infections,as anti-infectious agents for treating bacterial, fungal, and viralinfections. Among the disorders, diseases and conditions that thecompounds and pharmaceutical compositions of the present invention canbe used to treat or prevent are: age-associated memory impairment(AAMI), mild cognitive impairment (MCI), age-related cognitive decline(ARCD), pre-senile dementia, early onset Alzheimer's disease, seniledementia, dementia of the Alzheimer's type, Alzheimer's disease,cognitive impairment no dementia (CIND), Lewy body dementia,HIV-dementia, AIDS dementia complex, vascular dementia, Down syndrome,head trauma, traumatic brain injury (TBI), dementia pugilistica,Creutzfeld-Jacob Disease and prion diseases, stroke, ischemia, attentiondeficit disorder, attention deficit hyperactivity disorder, dyslexia,schizophrenia, schizophreniform disorder, schizoaffective disorder,cognitive dysfunction in schizophrenia, cognitive deficits inschizophrenia, Parkinsonism including Parkinson's disease,postencephalitic parkinsonism, parkinsonism-dementia of Gaum,frontotemporal dementia Parkinson's Type (FTDP), Pick's disease,Niemann-Pick's Disease, Huntington's Disease, Huntington's chorea,tardive dyskinesia, hyperkinesia, progressive supranuclear palsy,progressive supranuclear paresis, restless leg syndrome,Creutzfeld-Jakob disease, multiple sclerosis, amyotrophic lateralsclerosis (ALS), motor neuron diseases (MND), multiple system atrophy(MSA), corticobasal degeneration, Guillain-Barré Syndrome (GBS), andchronic inflammatory demyelinating polyneuropathy (CIDP), epilepsy,autosomal dominant nocturnal frontal lobe epilepsy, mania, anxiety,depression, premenstrual dysphoria, panic disorders, bulimia, anorexia,narcolepsy, excessive daytime sleepiness, bipolar disorders, generalizedanxiety disorder, obsessive compulsive disorder, rage outbursts,oppositional defiant disorder, Tourette's syndrome, autism, drug andalcohol addiction, tobacco addiction, obesity, cachexia, psoriasis,lupus, acute cholangitis, aphthous stomatitis, ulcers, asthma,ulcerative collitis, inflammatory bowel disease, Crohn's disease,spastic dystonia, diarrhea, constipation, pouchitis, viral pneumonitis,arthritis (including rheumatoid arthritis and osteoarthritis),endotoxaemia, sepsis, atherosclerosis, idiopathic pulmonary fibrosis,acute pain, chronic pain, neuropathies, urinary incontinence, diabetesand neoplasias.

Cognitive impairments or dysfunctions may be associated with psychiatricdisorders or conditions, such as schizophrenia and other psychoticdisorders, including but not limited to psychotic disorder,schizophreniform disorder, schizoaffective disorder, delusionaldisorder, brief psychotic disorder, shared psychotic disorder, andpsychotic disorders due to a general medical conditions, dementias andother cognitive disorders, induding but not limited to mild cognitiveimpairment, pre-senile dementia, Alzheimer's disease, senile dementia,dementia of the Alzheimer's type, age-related memory impairment, Lewybody dementia, vascular dementia, AIDS dementia complex, dyslexia,Parkinsonism including Parkinson's disease, cognitive impairment anddementia of Parkinson's Disease, cognitive impairment of multiplesclerosis, cognitive impairment caused by traumatic brain injury,dementias due to other general medical conditions, anxiety disorders,including but not limited to panic disorder without agoraphobia, panicdisorder with agoraphobia, agoraphobia without history of panicdisorder, specific phobia, social phobia, obsessive-compulsive disorder,post-traumatic stress disorder, acute stress disorder, generalizedanxiety disorder and generalized anxiety disorder due to a generalmedical condition, mood disorders, including but not limited to majordepressive disorder, dysthymic disorder, bipolar depression, bipolarmania, bipolar I disorder, depression associated with manic, depressiveor mixed episodes, bipolar II disorder, cyclothymic disorder, and mooddisorders due to general medical conditions, sleep disorders, includingbut not limited to dyssomnia disorders, primary insomnia, primaryhypersomnia, narcolepsy, parasomnia disorders, nightmare disorder, sleepterror disorder and sleepwalking disorder, mental retardation, learningdisorders, motor skills disorders, communication disorders, pervasivedevelopmental disorders, attention-deficit and disruptive behaviordisorders, attention deficit disorder, attention deficit hyperactivitydisorder, feeding and eating disorders of infancy, childhood, or adults,tic disorders, elimination disorders, substance-related disorders,including but not limited to substance dependence, substance abuse,substance intoxication, substance withdrawal, alcohol-related disorders,amphetamine or amphetamine-like-related disorders, caffeine-relateddisorders, cannabis-related disorders, cocaine-related disorders,halludnogen-related disorders, inhalant-related disorders,nicotine-related disorders, opioid-related disorders, phencyclidine orphencyclidine-like-related disorders, and sedative-, hypnotic- oranxiolytic-related disorders, personality disorders, including but notlimited to obsessive-compulsive personality disorder and impulse-controldisorders.

Cognitive performance may be assessed with a validated cognitive scale,such as, for example, the cognitive subscale of the Alzheimer's DiseaseAssessment Scale (ADAS-cog). One measure of the effectiveness of thecompounds of the present invention in improving cognition may includemeasuring a patient's degree of change according to such a scale.

The above conditions and disorders are discussed in further detail, forexample, in the American Psychiatric Association: Diagnostic andStatistical Manual of Mental Disorders, Fourth Edition, Text Revision,Washington, D.C., American Psychiatric Association, 2000. This Manualmay also be referred to for greater detail on the symptoms anddiagnostic features associated with substance use, abuse, anddependence.

Inflammation

The nervous system, primarily through the vagus nerve, is known toregulate the magnitude of the innate immune response by inhibiting therelease of macrophage tumor necrosis factor (TNF). This physiologicalmechanism is known as the “cholinergic anti-inflammatory pathway” (see,for example, Tracey, “The inflammatory reflex,” Nature 420: 853-9(2002)). Excessive inflammation and tumor necrosis factor synthesiscause morbidity and even mortality in a variety of diseases. Thesediseases include, but are not limited to, endotoxemia, rheumatoidarthritis, osteoarthritis, psoriasis, asthma, atherosclerosis,idiopathic pulmonary fibrosis, and inflammatory bowel disease.

Inflammatory conditions that can be treated or prevented byadministering the compounds described herein include, but are notlimited to, chronic and acute inflammation, psoriasis, endotoxemia,gout, acute pseudogout, acute gouty arthritis, arthritis, rheumatoidarthritis, osteoarthritis, allograft rejection, chronic transplantrejection, asthma, atherosclerosis, mononuclear-phagocyte dependent lunginjury, idiopathic pulmonary fibrosis, atopic dermatitis, chronicobstructive pulmonary disease, adult respiratory distress syndrome,acute chest syndrome in sickle cell disease, inflammatory bowel disease,Crohn's disease, ulcerative colitis, acute cholangitis, aphteousstomatitis, pouchitis, glomerulonephritis, lupus nephritis, thrombosis,and graft vs. host reaction.

Inflammatory Response Associated with Bacterial and/or Viral Infection

Many bacterial and/or viral infections are associated with side effectsbrought on by the formation of toxins, and the body's natural responseto the bacteria or virus and/or the toxins. The body's response toinfection often involves generating a significant amount of TNF and/orother cytokines. The over-expression of these cytokines can result insignificant injury, such as septic shock (when the bacteria is sepsis),endotoxic shock, urosepsis and toxic shock syndrome.

Cytokine expression is mediated by NNRs, and can be inhibited byadministering agonists or partial agonists of these receptors. Thosecompounds described herein that are agonists or partial agonists ofthese receptors can therefore be used to minimize the inflammatoryresponse associated with bacterial infection, as well as viral andfungal infections. Examples of such bacterial infections includeanthrax, botulism, and sepsis. Some of these compounds may also haveantimicrobial properties.

These compounds can also be used as adjunct therapy in combination withexisting therapies to manage bacterial, viral and fungal infections,such as antibiotics, antivirals and antifungals. Antitoxins can also beused to bind to toxins produced by the infectious agents and allow thebound toxins to pass through the body without generating an inflammatoryresponse. Examples of antitoxins are disclosed, for example, in U.S.Pat. No. 6,310,043 to Bundle et al. Other agents effective againstbacterial and other toxins can be effective and their therapeutic effectcan be complemented by co-administration with the compounds describedherein.

Pain

The compounds can be administered to treat and/or prevent pain, indudingacute, neurologic, inflammatory, neuropathic and chronic pain. Theanalgesic activity of compounds described herein can be demonstrated inmodels of persistent inflammatory pain and of neuropathic pain,performed as described in U.S. Published Patent Application No.20010056084 A1 (Allgeier et al.) (e.g., mechanical hyperalgesia in thecomplete Freund's adjuvant rat model of inflammatory pain and mechanicalhyperalgesia in the mouse partial sciatic nerve ligation model ofneuropathic pain).

The analgesic effect is suitable for treating pain of various genesis oretiology, in particular in treating inflammatory pain and associatedhyperalgesia, neuropathic pain and associated hyperalgesia, chronic pain(e.g., severe chronic pain, post-operative pain and pain associated withvarious conditions including cancer, angina, renal or biliary colic,menstruation, migraine and gout). Inflammatory pain may be of diversegenesis, including arthritis and rheumatoid disease, teno-synovitis andvasculitis. Neuropathic pain includes trigeminal or herpetic neuralgia,diabetic neuropathy pain, causalgia, low back pain and deafferentationsyndromes such as brachial plexus avulsion.

Neovascularization

The α7 NNR is associated with neovascularization. Inhibition ofneovascularization, for example, by administering antagonists (or atcertain dosages, partial agonists) of the α7 NNR can treat or preventconditions characterized by undesirable neovascularization orangiogenesis. Such conditions can include those characterized byinflammatory angiogenesis and/or ischemia-induced angiogenesis.Neovascularization associated with tumor growth can also be inhibited byadministering those compounds described herein that function asantagonists or partial agonists of α7 NNR.

Specific antagonism of α7 NNR-specific activity reduces the angiogenicresponse to inflammation, ischemia, and neoplasia. Guidance regardingappropriate animal model systems for evaluating the compounds describedherein can be found, for example, in Heeschen, C. et al., “A novelangiogenic pathway mediated by non-neuronal nicotinic acetylcholinereceptors,” J. Clin. Invest. 110(4):527-36 (2002).

Representative tumor types that can be treated using the compoundsdescribed herein include NSCLC, ovarian cancer, pancreatic cancer,breast carcinoma, colon carcinoma, rectum carcinoma, lung carcinoma,oropharynx carcinoma, hypopharynx carcinoma, esophagus carcinoma,stomach carcinoma, pancreas carcinoma, liver carcinoma, gallbladdercarcinoma, bile duct carcinoma, small intestine carcinoma, urinary tractcarcinoma, kidney carcinoma, bladder carcinoma, urothelium carcinoma,female genital tract carcinoma, cervix carcinoma, uterus carcinoma,ovarian carcinoma, choriocarcinoma, gestational trophoblastic disease,male genital tract carcinoma, prostate carcinoma, seminal vesiclescarcinoma, testes carcinoma, germ cell tumors, endocrine glandcarcinoma, thyroid carcinoma, adrenal carcinoma, pituitary glandcarcinoma, skin carcinoma, hemangiomas, melanomas, sarcomas, bone andsoft tissue sarcoma, Kaposi's sarcoma, tumors of the brain, tumors ofthe nerves, tumors of the eyes, tumors of the meninges, astrocytomas,gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas,Schwannomas, meningiomas, solid tumors arising from hematopoieticmalignancies (such as leukemias, chloromas, plasmacytomas and theplaques and tumors of mycosis fungoides and cutaneous T-celllymphoma/leukemia), and solid tumors arising from lymphomas.

The compounds can also be administered in conjunction with other formsof anti-cancer treatment, including co-administration withantineoplastic antitumor agents such as cis-platin, adriamycin,daunomycin, and the like, and/or anti-VEGF (vascular endothelial growthfactor) agents, as such are known in the art.

The compounds can be administered in such a manner that they aretargeted to the tumor site. For example, the compounds can beadministered in microspheres, microparticles or liposomes conjugated tovarious antibodies that direct the microparticles to the tumor.Additionally, the compounds can be present in microspheres,microparticles or liposomes that are appropriately sized to pass throughthe arteries and veins, but lodge in capillary beds surrounding tumorsand administer the compounds locally to the tumor. Such drug deliverydevices are known in the art.

Other Disorders

In addition to treating CNS disorders, inflammation, and undesirableneovascularization, and pain, the compounds of the present invention canbe also used to prevent or treat certain other conditions, diseases, anddisorders, in which NNRs play a role. Examples include autoimmunedisorders such as Lupus, disorders associated with cytokine release,cachexia secondary to infection (e.g., as occurs in AIDS, AIDS relatedcomplex and neoplasia), obesity, pemphitis, urinary incontinence,retinal diseases, infenctious diseases, myasthenia, Eaton-Lambertsyndrome, hypertension, osteoporosis, vasoconstriction, vasodilatation,cardiac arrhythmias, type I diabetes, bulimia, anorexia as well as thoseindications set forth in published PCT application WO 98/25619. Thecompounds of this invention can also be administered to treatconvulsions such as those that are symptomatic of epilepsy, and to treatconditions such as syphillis and Creutzfeld-Jakob disease.

Diagnostic Uses

The compounds can be used in diagnostic compositions, such as probes,particularly when they are modified to include appropriate labels. Theprobes can be used, for example, to determine the relative number and/orfunction of specific receptors, particularly the α7 receptor subtype.For this purpose the compounds of the present invention most preferablyare labeled with a radioactive isotopic moiety such as ¹¹C, ¹⁸F, ⁷⁶Br,¹²³I or ¹²⁵I.

The administered compounds can be detected using known detection methodsappropriate for the label used. Examples of detection methods includeposition emission topography (PET) and single-photon emission computedtomography (SPECT). The radiolabels described above are useful in PET(e.g., ¹¹C, ¹⁸F or ⁷⁶Br) and SPECT (e.g., ¹²³I) imaging, with half-livesof about 20.4 minutes for ¹¹C, about 109 minutes for ¹⁸F, about 13 hoursfor ¹²³I, and about 16 hours for ⁷⁶Br. A high specific activity isdesired to visualize the selected receptor subtypes at non-saturatingconcentrations. The administered doses typically are below the toxicrange and provide high contrast images. The compounds are expected to becapable of administration in non-toxic levels. Determination of dose iscarried out in a manner known to one skilled in the art of radiolabelimaging. See, for example, U.S. Pat. No. 5,969,144 to London of al.

The compounds can be administered using known techniques. See, forexample, U.S. Pat. No. 5,969,144 to London et al. The compounds can beadministered in formulation compositions that incorporate otheringredients, such as those types of ingredients that are useful informulating a diagnostic composition. Compounds useful in accordancewith carrying out the present invention most preferably are employed informs of high purity. See, U.S. Pat. No. 5,853,696 to Elmalch et al.

After the compounds are administered to a subject (e.g., a humansubject), the presence of that compound within the subject can be imagedand quantified by appropriate techniques in order to indicate thepresence, quantity, and functionality of selected NNR subtypes. Inaddition to humans, the compounds can also be administered to animals,such as mice, rats, dogs, and monkeys. SPECT and PET imaging can becarried out using any appropriate technique and apparatus. SeeVillemagne et al., In: Arneric et al. (Eds.) Neuronal NicotinicReceptors: Pharmacology and Therapeutic Opportunities, 235-250 (1998)and U.S. Pat. No. 5,853,696 to Elmalch et al.

The radiolabeled compounds bind with high affinity to selective NNRsubtypes (e.g., α7) and preferably exhibit negligible non-specificbinding to other nicotinic cholinergic receptor subtypes (e.g., α4β2 andthose receptor subtypes associated with muscle and ganglia). As such,the compounds can be used as agents for noninvasive imaging of nicotiniccholinergic receptor subtypes within the body of a subject, particularlywithin the brain for diagnosis associated with a variety of CNS diseasesand disorders.

In one aspect, the diagnostic compositions can be used in a method todiagnose disease in a subject, such as a human patient. The methodinvolves administering to that patient a detectably labeled compound asdescribed herein, and detecting the binding of that compound to selectedNNR subtypes (e.g., α7 receptor subtypes). Those skilled in the art ofusing diagnostic tools, such as PET and SPECT, can use the radiolabeledcompounds described herein to diagnose a wide variety of conditions anddisorders, including conditions and disorders associated withdysfunction of the central and autonomic nervous systems. Such disordersinclude a wide variety of CNS diseases and disorders, includingAlzheimer's disease, Parkinson's disease, and schizophrenia. These andother representative diseases and disorders that can be evaluatedinclude those that are set forth in U.S. Pat. No. 5,952,339 to Bencherifet al.

In another aspect, the diagnostic compositions can be used in a methodto monitor selective nicotinic receptor subtypes of a subject, such as ahuman patient. The method involves administering a detectably labeledcompound as described herein to that patient and detecting the bindingof that compound to selected nicotinic receptor subtypes namely, the α7receptor subtype.

Receptor Binding

The compounds of this invention can be used as reference ligands inbinding assays for compounds which bind to NNR subtypes, particularlythe α7 receptor subtype. For this purpose the compounds of thisinvention are preferably labeled with a radioactive isotopic moiety suchas ³H, or ¹⁴C. Examples of such binding assays are described in detailbelow.

Pharmaceutical Compositions

Although it is possible to administer the compound of the presentinvention in the form of a bulk active chemical, it is preferred toadminister the compound in the form of a pharmaceutical composition orformulation. Thus, one aspect the present invention includespharmaceutical compositions comprising the compound of the presentinvention and one or more pharmaceutically acceptable carriers,diluents, or excipients. Another aspect of the invention provides aprocess for the preparation of a pharmaceutical composition indudingadmixing the compound of the present invention with one or morepharmaceutically acceptable carriers, diluents or excipients.

The manner in which the compound of the present invention isadministered can vary. The compound of the present invention ispreferably administered orally. Preferred pharmaceutical compositionsfor oral administration include tablets, capsules, caplets, syrups,solutions, and suspensions. The pharmaceutical compositions of thepresent invention may be provided in modified release dosage forms suchas time-release tablet and capsule formulations.

The pharmaceutical compositions can also be administered via injection,namely, intravenously, intramuscularly, subcutaneously,intraperitoneally intraarterially, intrathecally, andintracerebroventricularly. Intravenous administration is a preferredmethod of injection. Suitable carriers for injection are well known tothose of skill in the art and include 5% dextrose solutions, saline, andphosphate buffered saline.

The formulations may also be administered using other means, forexample, rectal administration. Formulations useful for rectaladministration, such as suppositories, are well known to those of skillin the art. The compounds can also be administered by inhalation, forexample, in the form of an aerosol; topically, such as, in lotion form;transdermally, such as, using a transdermal patch (for example, by usingtechnology that is commercially available from Novartis and AlzaCorporation), by powder injection, or by buccal, sublingual, orintranasal absorption.

Pharmaceutical compositions may be formulated in unit dose form, or inmultiple or subunit doses

The administration of the pharmaceutical compositions described hereincan be intermittent, or at a gradual, continuous, constant or controlledrate. The pharmaceutical compositions may be administered to awarm-blooded animal, for example, a mammal such as a mouse, rat, cat,rabbit, dog, pig, cow, or monkey; but advantageously is administered toa human being. In addition, the time of day and the number of times perday that the pharmaceutical composition is administered can vary.

The compound of the present invention may be used in the treatment of avariety of disorders and conditions and, as such, may be used incombination with a variety of other suitable therapeutic agents usefulin the treatment or prophylaxis of those disorders or conditions. Thus,one embodiment of the present invention includes the administration ofthe compound of the present invention in combination with othertherapeutic compounds. For example, the compound of the presentinvention can be used in combination with other NNR ligands (such asvarenicline), antioxidants (such as free radical scavenging agents),antibacterial agents (such as penicillin antibiotics), antiviral agents(such as nucleoside analogs, like zidovudine and acyclovir),anticoagulants (such as warfarin), anti-inflammatory agents (such asNSAIDs), anti-pyretics, analgesics, anesthetics (such as used insurgery), acetylcholinesterase inhibitors (such as donepezil andgalantamine), antipsychotics (such as haloperidol, clozapine,olanzapine, and quetiapine), immuno-suppressants (such as cyclosporinand methotrexate), neuroprotective agents, steroids (such as steroidhormones), corticosteroids (such as dexamethasone, predisone, andhydrocortisone), vitamins, minerals, nutraceuticals, anti-depressants(such as imipramine, fluoxetine, paroxetine, escitalopram, sertraline,venlafaxine, and duloxetine), anxiolytics (such as alprazolam andbuspirone), anticonvulsants (such as phenytoin and gabapentin),vasodilators (such as prazosin and sildenafil), mood stabilizers (suchas valproate and aripiprazole), anti-cancer drugs (such asanti-proliferatives), antihypertensive agents (such as atenolol,clonidine, amiopidine, verapamil, and olmesartan), laxatives, stoolsofteners, diuretics (such as furosemide), anti-spasmotics (such asdicyclomine), anti-dyskinetic agents, and anti-ulcer medications (suchas esomeprazole). Such a combination of pharmaceutically active agentsmay be administered together or separately and, when administeredseparately, administration may occur simultaneously or sequentially, inany order. The amounts of the compounds or agents and the relativetimings of administration will be selected in order to achieve thedesired therapeutic effect. The administration in combination of acompound of the the present invention with other treatment agents may bein combination by administration concomitantly in: (1) a unitarypharmaceutical composition including both compounds; or (2) separatepharmaceutical compositions each including one of the compounds.Alternatively, the combination may be administered separately in asequential manner wherein one treatment agent is administered first andthe other second. Such sequential administration may be close in time orremote in time.

Another aspect of the present invention includes combination therapycomprising administering to the subject a therapeutically orprophylactically effective amount of the compound of the presentinvention and one or more other therapy including chemotherapy,radiation therapy, gene therapy, or immunotherapy.

EXAMPLES

The following examples are provided to illustrate the present invention,and should not be construed as limiting thereof. In these examples, allparts and percentages are by weight, unless otherwise noted.

Nuclear Magnetic Resonance (NMR) Spectrometry

NMR spectra were collected on either a Varian Unity 300 MHz instrumentor a Bruker 400 MHz instrument equipped with an auto-sampler andcontrolled by a DRX400 console. Automated experiments were acquiredusing ICONNMR v4.0.4 (build 1) running with Topspin v 1.3 (patch level8) using the standard Bruker loaded experiments. For non-routinespectroscopy, data were acquired through the use of Topspin alone.

Melting Point

A Fisher-Johns hot stage melting point apparatus was used, at a settingcorresponding to a heating rate of about 5° C. per min.

Differential Scanning Calorimetry (DSC)

Dsc data were collected on a Mettler DSC 823e equipped with a 50position auto-sampler. The instrument was calibrated for energy andtemperature using certified indium. Typically 0.5-1.5 mg of each sample,in a pin-holed aluminum pan, was heated at 10° C.min−1 from 25° C. to300° C. A nitrogen purge at 50 ml/min−1 was maintained over the sample.Instrument control and data analysis were performed using the stare v9.10 software package.

X-Ray Powder Diffraction (XRPD)

Method 1

X-Ray Powder Diffraction patterns were collected on a Siemens D5000diffractometer using Cu Kα radiation (40 kV, 40 mA), θ-θ goniometer,divergence of V20 and receiving slits, a graphite secondarymonochromator and a scintillation counter. The instrument is performancechecked using a certified Corundum standard (NIST 1976). The softwareused for data collection was Diffrac Plus XRD Commander v2.3.1 and thedata were analysed and presented using Diffrac Plus EVA v 11,0.0.2 or v13.0.0.2.

Samples were run under ambient conditions as flat plate specimens usingpowder as received. Approximately 30 mg of the sample was gently packedinto a cavity cut into polished, zerobackground (510) silicon wafer. Thesample was rotated in its own plane during analysis. The details of thedata collection are:

-   Angular range: 2 to 42°2θ-   Step size: 0.05°2θ or 0.1°2θ0-   Collection time: 4 s.step⁻¹    Method 2

X-Ray Powder Diffraction patterns were collected on a Bruker AXS C2GADDS diffractometer using Cu Kα radiation (40 kV, 40 mA), automated XYZstage, laser video microscope for auto-sample positioning and a HiStar2-dimensional area detector. X-ray optics consists of a single Göbelmultilayer mirror coupled with a pinhole collimator of 0.3 mm.

The beam divergence, i.e. the effective size of the X-ray beam on thesample, was approximately 4 mm. A θ-θ continuous scan mode was employedwith a sample—detector distance of 20 cm which gives an effective 2θrange of 3.2°-29.7°. Typically the sample would be exposed to the X-raybeam for 120 seconds. The software used for data collection was GADDSfor WNT 4.1.16 and the data were analysed and presented using DiffracPlus EVA v 9.0.0.2 or v 13.0.0.2.

Samples run under ambient conditions were prepared as flat platespecimens using powder as received without grinding. Approximately 1-2mg of the sample was lightly pressed on a glass slide to obtain a flatsurface. Samples run under non-ambient conditions were mounted on asilicon wafer with heatconducting compound. The sample was then heatedto the appropriate temperature at ca. 10° C.min⁻¹ and subsequently heldisothermally for ca 1 minute before data collection was initiated.

Single Crystal X-Ray Diffraction (SCXD)

Data were collected on a Bruker AXS 1K SMART CCD diffractometer equippedwith an Oxford Cryosystems Cryostream cooling device. Structures weresolved using either the SHELXS or SHELXD programs and refined with theSHELXL program as part of the Bruker AXS SHELXTL suite. Unless otherwisestated, hydrogen atoms attached to carbon were placed geometrically andallowed to refine with a riding isotropic displacement parameter.Hydrogen atoms attached to a heteroatom were located in a differenceFourier synthesis and were allowed to refine freely with an isotropicdisplacement parameter.

Example 1 Synthesis of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide

To a suspension of(2S,3R)-3-amino-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]octane (20mg, 0.092 mmol, prepared as described in PCT WO 09/018505, hereinincorporated by reference with regard to such synthesis),o-(benzotriazol-1-yl)-N,N,N,1-tetramethyluronium hexafluorophosphate(HBTU, 41.7 mg, 0.110 mmol) and 3,5-difluorobenzoic acid (17.4 mg, 0.110mmol) in N,N-dimethylformamide (DMF, 2 ml) was added triethylamine (28mg, 0.28 mmol) at room temperature. The reaction mixture was stirredovernight at room temperature, diluted with ethyl acetate (200 ml) andwashed with 20% aqueous potassium carbonate. The residue was purified bysilica gel chromatography with the eluent methanol:triethylamine=300:1.The solvent was removed to give(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(30 mg, 76%), purity by HPLC: 100% (214 nm), 98.2% (254 nm); ¹H NMR (400MHz, CDCl₃) d 8.48 (d, j=2.0 Hz, 1H), 8.36 (dd, j=1.7 Hz, j=4.9 Hz, 1H),7.56-7.60 (m, 1H), 7.14-7.20 (m, 1H), 7.05-7.14 (m, 2H), 6.87-6.96 (m,1H), 6.23 (d, j=7.8 Hz, 1H), 3.88-3.96 (m, 1H), 3.02-3.13 (m, 1H),2.82-3.00 (m, 4H), 2.65-2.82 (m, 2H), 1.97-2.05 (m, 1H), 1.58-1.84 (m,3H), 1.43-1.55 (m, 1H); ESI-MS 358.1(MH)⁺.

Example 2 Synthesis of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidefumarate

To a solution of 3,5-difluorobenzoic acid (9.36 g, 59.2 mmol),chloroform (200 mL) and triethylamine (16.34 g, 161.5 mmol) at 25° C.was added HBTU (22.5 g, 59.2 mmol). The mixture was heated to 40-42° C.for 45 min resulting in the formation of a white suspension. Thesuspension was cooled to 10° C. and a solution of(2S,3R)-3-amino-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]octane (11.7g, 53.8 mmoles) in chloroform (50 mL) was added over a 15-20 min periodand stirred for 1.5 h. The reaction mixture was heated to 40-42° C. andadditional 3,5-difluorobenzoic acid (2.0 g, 13 mmol) and HBTU (4 g, 11mmol) were added, followed by stirring at 40-42° C. for 2 h and then atambient temperature for 16 h. The reaction mixture was quickly quenchedwith water (200 mL), and under stirring, the pH of the aqueous layer wasadjusted to pH=10-11 with 10 wt % aqueous sodium hydroxide. The layerswere separated and the organic layer was washed twice with water (100mL). The solvent was removed in vacuo to afford 22.9 g of a viscousorange solid. The strength of the product in the crude oil wasdetermined at 66.0 wt % by quantitative HPLC against a referencestandard; this corresponds to a yield of 15.1 g (78%). The oil wasdissolved in methyl ethyl ketone (50 mL) which was subsequentlydistilled off in vacuo; this process was repeated a total of threetimes. A 500 mL three-necked, round-bottomed flask, equipped with anoverhead stirrer, temperature probe, dropping funnel and condenser, wascharged with fumaric acid (4.9 g, 42 mmol) and methyl ethyl ketone (150mL). The suspension was heated to 78° C. which led to completedissolution of the acid. A solution of the(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidein methyl ethyl ketone (50 mL) was added slowly, keeping the internaltemperature above 75° C. After completion of the addition, thesuspension was stirred for 30-45 min at 78° C. and the heat source wasturned off. The suspension was stirred overnight, filtered and the cakewas dried at 50° C. in vacuo for 16 h to afford(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidefumarate as a light yellow, crystalline solid (99.6% pure by HPLC), mp208-210° C. Yield: 65% for two steps. ¹H NMR (D₂O, 400 MHz): δ 8.30 (d,J=5.6 Hz, 1H); 8.06 (d, J=7.5 Hz, 1H); 7.48 (dd, J=8.7 Hz, J=5.6 Hz, 1H); 6.97-7.06 (m, 1H); 6.75-6.85 (m, 2 H); 6.49 (s, 2H); 4.15 (d, J=7.5Hz, 1H); 3.69-3.81 (m, 1H); 3.40-3.59 (m, 2H); 3.17-3.40 (m, 4 H);2.03-2.18 (m, 2H); 1.91-2.03 (m, 2 H), 1.78-1.91 (m, 1H).

Example 3 Synthesis of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidemono-hydrochloride

Procedure A: To a solution of 250 mg (0.7 mmol) of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidein 10 mL of isopropyl acetate was added aqueous hydrochloric acid (65 μLof a 37% (w/w), 0.78 mmol). The solution was heated to 50° C. and cooledto 0° C. over a 4 h period. The mono-hydrochloride sample was a mixtureof gum and white powder at 0° C. The sample was then heated to 20° C.and cooled again to 0° C. (cooling ramp 5° C./min). The resulting solidswere collected and dried under vacuum at 25° C. for 24 h. mp(DSC)=274.8° C.

Procedure B: Concentrated hydrochloric acid (0.54 mL of 37% (w/w), 6.6mmol) was added drop-wise, with ice bath cooling, to tetrahydrofuran(THF , ˜4 mL) and diluted to 5 mL volume with THF. This solution wasadded drop-wise (over a 5 min period) to a warm (45-50° C.) solution of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(2.43 g of 96.8% purity, 6.58 mmol) in acetone (20 mL). Solids began toprecipitate. The mixture was heated near boiling for 15 min, cooled toambient temperature and allowed to sit 16 h. The solids were collectedby suction filtration under nitrogen, washed with acetone and dried in avacuum oven (85° C., 3 h). This left 2.26 g of material that was 93%pure by LCMS. The entire sample was digested in hot (near boiling)2-propanol (25 mL) for 10 min. The mixture was cooled to ambienttemperature and allowed to stand for 3 h. The solids were collected bysuction filtration under nitrogen and dried in a vacuum oven (85° C.,2.5 h). The resulting white crystals were >99% pure by HPLC, weighed2.03 g (78.4% yield) and melted at 273-276° C. ¹H NMR (400 MHz, DMSO-d₆)δ 10.45 (broad s, 1H), 8.66 (d, 1H), 8.54 (s, 1H), 8.28 (d, 1H), 7.76(d, 1H), 7.41 (m, 1H), 7.24 (m, 3H), 4.14 (m, 1H), 4.07 (m, 1H), 3.46(m, 2H), 3.10-3.35 (m, 4H), 2.06 (m, 3H), 1.90 (m, 1H), 1.69 (m, 1H).Elemental analysis: Calculated for C₂₀H₂₁ON₃F₂.HCl (C, 60.99%; H, 5.63%,N, 10.67%); Found (C, 60.94%, 60.91%; H, 5.64%, 5.66%; N, 10.63%,10.67%).

Example 4 Synthesis of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidemono-phosphate

To a solution of 250 mg (0.7 mmol) of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidein 5 mL of isopropyl alcohol was added 780 μL (0.78 mmol, 1.1 eq) of a1M phosphoric acid in THF solution. The solution was heated to 50° C.and cooled to 0° C. over a 4 hour period. A white immobile slurry formedat 0° C., which remained after warming the sample to room temperature.Evaporation of the solvent yielded crystalline material that wascollected and dried under vacuum at 25° C. for 24 h. mp (DSC)=219.2° C.¹H NMR (400 MHz, DMSO-d₆) δ 8.48 (s, 1H), 8.34 (d, 1H), 8.28 (d, 1H),7.68 (d, 1H), 7.43 (m, 1H), 7.24 (m, 3H), 5.04 (br s), 3.84 (m, 1H),2.70-3.35 (m, 7H), 1.60-1.90 (m, 4H), 1.40 (m, 1H).

Example 6 Synthesis of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azablcyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidemono-4-hydroxybenzoate

To a solution of 250 mg (0.70 mmol) of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidein 5 mL of isopropyl acetate was added 780 μL (0.78 mmol, 1.1 eq) of a1M 4-hydroxybenzoic acid in THF solution. The solution was heated to 50°C. and cooled to 0° C. over a 4 hour period. The mono-4-hydroxybenzoatewas obtained as a gum. The crystallization was obtained after seedingwith the hemi-4-hydroxybenzoate and 48 hours of maturation between 50°C. and room temperature (4 hours cycle) of an evaporated mixture of gumand solvent (only a quarter of the starting volume was remaining). Thesolid was then isolated by evaporation of the solvent under nitrogen.The resulting solids were collected and dried under vacuum at 25° C. for24 h. mp (DSC)=144.0° C.

Example 6 Synthesis of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidehemi-4-hydroxybenzoate

To a solution of 71.5 mg (0.20 mmol) of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidein 3.5 mL of isopropyl acetate was added 100 μL (0.1 mmol, 0.5 eq) of a1M 4-hydroxylbenzoic acid in THF solution. The isopropyl acetate wasevaporated to yield(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidemono-4-hydroxybenzoate as a solid which was collected and dried undervacuum at 25° C. for 24 h. mp(DSC)=106.0° C. ¹H NMR (400 MHz, DMSO-d₆) δ10.23 (br s), 8.43 (s, 1H), 8.29 (s, 1H), 8.28 (s, 1H), 7.78 (d, 1H),7.61 (m, 1H), 7.41 (m, 3H), 7.22 (m, 1H), 6.80 (d, 1H), 3.66 (m, 1H),2.70-3.20 (m, 7H), 1.50-1.90 (m, 4H), 1.20 (m, 1H).

Example 7 Synthesis of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidemonohydrate

To a solution of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(993 mg, 2.80 mmol) water (5 mL) was added chloroform (15 mL). The pH ofthe aqueous layer was adjusted to pH=10-11 with 10 weight % sodiumhydroxide. The biphasic mixture was shaken vigorously, and the layerswere allowed to separate. The chloroform layer was isolated, and theaqueous layer was extracted once more with chloroform (9 mL). Thecombined chloroform layers were washed once with water (7 mL), filteredover a bed of anhydrous magnesium sulfate and concentrated in vacuo toafford a colorless, clear oil with a tendency to foam. The material wastreated with methyl tert-butyl ether (MTBE, 10-15 mL) followed bysolvent distillation in vacuo; this process was repeated once more. Thematerial was dissolved in MTBE (10-15 mL) and heptane was added until awhite cloudiness appeared. At this point a slow distillation ofvolatiles at 50-55° C. at ambient pressure was started and additionalsolid material separated out. The distillation was halted and thematerial was collected by filtration and washed with a small amount ofheptane. The material was dried in vacuo at 55° C. under avacuum/nitrogen bleed for 60 h and at 70-85° C. for 40 h to afford 400mg of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidemono-hydrate as a white brittle solid: α_(D) ^(26.6° C.)=40°; elementalanalysis, calc: C (63.99); H (6.18); N (11.19), H₂O (4.8 weight %),found: C (64.23); H (6.27); N (11.18); H₂O (4.48). ¹H NMR (CDCl₃, 400MHz): δ 8.46 (d, J=2 Hz, 1H); 8.35 (dd, J=4.8 Hz, J=2 Hz, 1 H);7.56-7.61 (m, 1H); 7.08-7.19 (m, 3H); 6.87-6.95 (m, 1H), 6.32 (d, J=8.1Hz, 1H); 3.89-3.95 (m, 1H); 3.00-3.12 (m, 1H), 2.84-2.99 (m, 4H);2.68-2.84 (m, 2H); 1.98-2.04 (m, 1H); 1.83 (s, 2H); 1.58-1.79 (m, 3H);1.44-1.54 (m, 1H). Decomposes at 240° C.

Example 8 Synthesis of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidehemi-galactarate

To a stirred solution of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(357 mg, 1.00 mmol) in absolute ethanol (10 mL) at 70-72° C. was addedgalataric acid (neat) (105 mg, 0.50 mmol) in small portions. Heating wascontinued for an additional 15 min after completion of acid addition.The solution was slowly cooled to ambient temperature. After standingfor 2 h, the solids were collected by vacuum filtration, washed withethanol, and dried under a nitrogen cone for 30 min. The resultingmaterial was dried for 3 h at 75° C. in a vacuum oven to remove residualethanol. The results of XRPD analysis are shown in FIG. 11.

Example 9 Salt Screening of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide

Stock solutions of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidefree base were prepared as follows:

-   20 mg/ml in IPA, 25 mg/ml in i-ProAc—Counter-ions 1-12-   25 mg/ml in i-ProAc—Counter-ions 13-20

Each vial was charged with 2 ml of free base stock solution at ambient(40-50 mg of free base/vial). To each vial was then added appropriatevolumes of stock acid solution, (1M in THF, unless otherwise stated) ateither 1.1 or 2.2 equivalents at ambient. Insoluble acids were added assolids accordingly. All samples were the warmed to 50° C. prior tocooling to 0° C. over 10 hours. All amorphous solids, including gums andoils, were placed on maturation (ambient-50° C. in 4 hour cycles over 2days) followed by XRPD re-analysis. To those samples which remainedamorphous post maturation 2 ml of methyl ethyl ketone was added and thesamples further matured for 3 days. Clear solutions were sequentiallyevaporated to approximately to half and then to quarter volume at 50° C.Remaining solutions were further cooled to 5° C. prior to the completeremoval of the solvent under vacuum. All resulting solids were analyzedby XRPD and any crystalline samples with unique XRPD patterns wereanalyzed further by ¹HNMR/Ion chromatography, solid state stability at40° C./75% RH for 1 week and aqueous solubility (target 10 mg/ml at 25°C., unbuffered)

Acidic counter-ions selected for the salt selection study pKa Nr. AcidClass 1 2 3 LogP MW 1 Hydrochloric 1 −6.10 — 36.46 acid 37 wt % (12M) 2Sulphuric 1 −3.00 1.92 −1.03 98.08 acid 3 Methane 2 −1.20 −1.89 96.10sulfonic acid 4 Maleic acid 1 1.92 6.23 −0.01 116.07 5 Phosphoric 1 1.967.12 12.32 −2.15 98.00 acid 6 L-Tartaric 1 3.02 4.36 −1.43 150.09 acid 7Fumaric 1 3.03 4.38 −0.01 116.07 acid used as powder 8 Citric acid 13.13 4.76 6.40 −1.72 192.12 9 L-Malic acid 1 3.46 5.10 −1.26 134.09 101-Hydroxy- 2 2.70 13.50 3.29 188.17 2-Naphthoic acid used as powder 114-Hydroxy 2 benzoic acid 12 Succinic acid 1 4.21 5.64 −0.59 118.09 1M inMeOH 13 Benzene 2 0.70 0.47 158.18 sulfonic acid 14 p-Toluene 2 −1.340.93 190.22 sulfonic acid 1M in EtOH 15 Hippuric acid 1 3.55 0.31 179.17used as powder 16 D-Gluconic 1 3.76 −3.18 196.16 acid 50% in water 17Acetic acid 1 4.76 −0.29 60.05 18 Benzoic acid 2 4.19 1M in IPA 19Propionic 2 4.87 0.25 74.07 acid 20 L-Aspartic 1 1.88 3.65 −0.67 133.11acid used as powder

TABLE Salt screen summary XRPD after Target Observation on XRPD XRPDafter evaporation and Observation Counter- Stoichi- addition of acidObservation analysis 48 hours of maturation on 2nd ion Solvent ometry atRT at 0° C. after filtration maturation in MEK maturation Hydro-2-propanol mono salt Clear solution White powder Crystalline 

Crystalline 

n/a n/a chloride bis salt Clear solution Clear solution n/a Clearsolution Crystalline 

n/a Isopropyl mono salt Precipitate White powder Crystalline 

Crystalline 

n/a n/a acetate bis salt Precipitate White powder gum 

Crystalline 

n/a n/a Sulphate 2-propanol mono salt Precipitate White powder gum 

Crystalline 

n/a n/a (Sulfate) bis salt Precipitate gum gum 

gum gum gum Isopropyl mono salt Precipitate White powder Amor-Amorphous 

Amorphous 

White powder acetate phous 

bis salt Precipitate White powder gum 

Clear solution gum 

gum 

Mesylate 2-propanol mono salt Clear solution Clear solution n/a n/a n/an/a bis salt Clear solution Clear solution n/a Amorphous 

Amorphous 

White powder Isopropyl mono salt Precipitate gum n/a Crystalline 

n/a n/a acetate bis salt Precipitate gum n/a Crystalline 

n/a n/a Maleate 2-propanol mono salt Clear solution Clear solution n/aClear solution n/a gum bis salt Clear solution Clear solution n/a Clearsolution n/a gum Isopropyl mono salt gum gum n/a gum 

n/a gum acetate bis salt gum gum n/a gum 

n/a gum Phosphate 2-propanol mono salt Precipitate White powder n/aCrystalline 

n/a n/a Not enough material bis salt Precipitate White powder gum 

White powder Amorphous 

White powder Isopropyl mono salt Precipitate gum n/a Crystalline 

n/a n/a acetate bis salt Precipitate gum n/a Crystalline 

n/a n/a Fumarate 2-propanol mono salt Clear solution Clear solution n/aClear solution Crystalline n/a bis salt Stopped experiment♥ Isopropylmono salt Clear solution Precipitate Mainly Crystalline 

n/a n/a acetate amorphous 

bis salt Clear solution Precipitate Amor- Crystalline 

n/a n/a phous 

Citrate 2-propanol mono salt Precipitate White powder gum 

n/a gum 

gum 

bis salt Precipitate White powder gum 

n/a gum 

gum 

Isopropyl mono salt Precipitate White powder Amor- Amorphous 

Amorphous 

White powder acetate phous 

bis salt Precipitate White powder Amor- Amorphous 

Amorphous 

White powder phous 

Malate 2-propanol mono salt Clear solution Clear solution n/a Clearsolution gum 

gum 

bis salt Clear solution Clear solution n/a Clear solution gum 

gum 

Isopropyl mono salt Precipitate White powder Amor- Amorphous Amorphous 

White powder acetate phous 

bis salt Precipitate White powder gum 

Low n/a n/a Crystallinity 

Xinafoate 2-propanol mono salt Clear solution Clear solution n/a Clearsolution gum 

gum 

bis salt Clear solution Clear solution n/a Clear solution gum 

gum 

Isopropyl mono salt Clear solution Clear solution n/a Clear solutiongum 

gum 

acetate bis salt Clear solution Clear solution n/a Clear solution gum 

gum 

4-Hydroxy- 2-propanol mono salt Clear solution Clear solution n/a Clearsolution gum 

gum 

benzoate bis salt Clear solution Clear solution n/a Clear solution gum 

gum 

Isopropyl mono salt Clear solution Clear solution n/a Crystalline 

n/a n/a acetate bis salt Clear solution Clear solution n/a Clearsolution gum 

gum 

Succinate 2-propanol mono salt Clear solution Clear solution n/a Clearsolution gum 

gum 

bis salt Clear solution Clear solution n/a Clear solution gum 

gum 

Isopropyl mono salt Clear solution Clear solution n/a Clear solutiongum 

gum 

acetate bis salt Clear solution Clear solution n/a Clear solution gum 

gum 

XRPD after Observation evaporation Observa- XRPD post Target on XRPDXRPD after 48 and tion evaporation Counter- Stoichi- addition ofObservation analysis hours of maturation on 2nd and storage ion Solventometry acid at RT at 0° C. after filtration maturation in MEK maturationat 5° C. Benzylate Isopropyl mono salt Precipitate White powder Amor-Low n/a n/a n/a Acetate phous 

Crystallinity 

Tosylate Isopropyl mono salt Clear solution Clear solution n/a Clearsolution Crystal- n/a n/a Acetate line 

Hippurate Isopropyl mono salt Clear solution Clear solution n/a Clearsolution gum 

gum 

n/a Acetate Gluconate Isopropyl mono salt Clear solution Clear solutionn/a Clear solution gum 

gum 

n/a Acetate Acetate Isopropyl mono salt Clear solution Clear solutionn/a Clear solution gum 

gum 

n/a Acetate Benzoate Isopropyl mono salt Clear solution Clear solutionn/a Clear solution gum 

gum 

Crystalline 

Acetate Propionate Isopropyl mono salt Clear solution Clear solution n/aClear solution gum 

gum 

n/a Acetate Aspartate Isopropyl mono salt Non-dissolved Non-dissolvedCrystalline Crystalline Crystalline n/a n/a Acetate same as the same asthe same as the acid♥ acid♥ acid♥ Key:

Crystalline

Amorphous ♥No further analysis performed n/a Not applicable

TABLE Characterization after primary salt screening XRPD Stoichiometry(Acid:Base) 40 C./75% Target XRPD Ion Chroma- RH 1 Week Aqueous SaltStoichiometry after filtration 1H NMR tography (See FIG. 12) SolubilityHydrochloride mono salts Pattern1 Confirmation of the salt formation1.0:1 Pattern1 

>10 mg/mL bis salts Pattern2 Confirmation of the salt formation n/adeliquescent 

n/a Pattern3 Phosphate mono salts Pattern1 Confirmation of the saltformation 1.0:1 Pattern1 

>10 mg/mL mono and Pattern2 Confirmation of the salt formation n/aPattern1 

+ >10 mg/mL bis salts Pattern2 

Fumarate mono salt Pattern1 Confirmation of the formation n/a Pattern1 

>10 mg/mL of a mono salt 1.0:1 bis salt Pattern2 Confirmation of theformation n/a Pattern2 

>10 mg/mL of a bis salt 1.9:1 4-Hydroxybenzoate mono salt Pattern1Confirmation of the formation n/a Pattern1 

>10 mg/mL of a hemi salt 0.5:1 Benzoate mono salt Pattern1 Confirmationof mono salt formation n/a Pattern2 

0.8 mg/mL Sulphate mono salt Pattern1 Confirmation of the salt formationn/a deliquescent 

n/a Mesylate mono and Pattern1 Confirmation of the formation n/adeliquescent 

n/a bis salts of a mono salt Malate bis salts Pattern1 Confirmation ofthe salt formation 1.4:1 n/a deliquescent 

n/a Low crystallinity Tosylate mono salts Pattern1 Confirmation of thesalt formation 1:1 n/a deliquescent 

n/a Key:

Crystalline

Amorphous ♥No further analysis performed n/a Not applicable

Example 10 Crystal Structure of the Hydrochloride Salt

Crystals of hydrochloride salt were obtained by maturation between roomtemperature and 50° C. of a methanol solution of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidemono-hydrochloride. The single crystal structure data are indicated inthe table below. The sample was checked to be representative of the bulk

Single Crystal Structure of the mono hydrochloride salt Molecularformula C₂₀H₂₂ClF₂N₃O Molecular weight 393.86 Crystal system MonoclinicSpace group P2₁ a 10.049(1) Å α 90° b 8.872(1) Å β 94.088(3)° c10.491(1) Å γ 90° V 933.07(1) Å³ Z 2 D_(c) 1.402 g · cm⁻¹ μ 0.239 mm⁻¹Source, λ Mo—K(alpha), 0.71073 Å F(000) 412 T 120(2)K Crystal colourlessprism, 0.3 × 0.15 × 0.11 mm Data truncated to 0.80 Å θ_(max) 26.37°Completeness 99.4% Reflections 7986 Unique reflections 3750 R_(int)0.0135 Flack parameter −0.04(3) R_(all) 0.0236 R₁ 0.0231

The structure solution was obtained by direct methods, full-matrixleast-squares refinement on F² with weighting W⁻¹=σ²(F_(o) ²)+(0.0435P)²+(0.1500 P), where P=(F_(o) ²+2F_(c) ²)/3, anisotropic displacementparameters, empirical absorption corrections were applied, absolutestructure parameter=−0.04(3). Final wR²={Σ[w(F_(o) ²−F_(c) ²)²]/Σ[w(F₀²)²]^(1/2)}=0.0636 for all data, conventional R₁=0.0231 on F values of3684 reflections with F_(o)>4σ(F_(o)), S=1.004 for all data and 252parameters. Final Δ/σ(max) 0.001, Δ/σ(mean), 0.000. Final difference mapbetween +0.195 and −0.136 e Å⁻³.

The value of the absolute structure parameter enabled the determinationof the configuration of the chiral centers. This configuration isindicated in FIG. 10.

Example 11 Biological Assays Radioligand Binding at CNS nAChRs α4β2 NNRSubtype

Preparation of membranes from rat cortex: Rats (female, Sprague-Dawley),weighing 150-250 g, were maintained on a 12 h light/dark cycle and wereallowed free access to water and food supplied by PMI NutritionInternational, Inc. Animals were anesthetized with 70% CO₂, and thendecapitated. Brains were removed and placed on an ice-cold platform. Thecerebral cortex was removed and placed in 20 volumes (weight:volume) ofice-cold preparative buffer (137 mM NaCl, 10.7 mM KCI, 5.8 mM KH₂PO₄, 8mM Na₂HPO₄, 20 mM HEPES (free acid), 5 mM iodoacetamide, 1.6 mM EDTA, pH7.4); PMSF, dissolved in methanol to a final concentration of 100 μM,was added and the suspension was homogenized by Polytron. The homogenatewas centrifuged at 18,000× g for 20 min at 4° C. and the resultingpellet was re-suspended in 20 volumes of ice-cold water. After 60 minincubation on ice, a new pellet was collected by centrifugation at18,000× g for 20 min at 4° C. The final pellet was re-suspended in 10volumes of buffer and stored at −20° C.

Preparation of membranes from SH-EP1/human α4β2 clonal cells: Cellpellets from 40 150 mm culture dishes were pooled, and homogenized byPolytron (Kinematica GmbH, Switzerland) in 20 milliliters of ice-coldpreparative buffer. The homogenate was centrifuged at 48,000 g for 20minutes at 4° C. The resulting peUet was re-suspended in 20 mL ofice-cold preparative buffer and stored at −20° C.

On the day of the assay, the frozen membranes were thawed and spun at48,000× g for 20 min. The supernatant was decanted and discarded. Thepellet was resuspended in Dulbecco's phosphate buffered saline (PBS,Life Technologies) pH 7.4 and homogenized with the Polytron for 6seconds. Protein concentrations were determined using a Pierce BCAProtein Assay Kit, with bovine serum albumin as the standard (PierceChemical Company, Rockford, Ill.).

Membrane preparations (approximately 50 μg for human and 200-300 μgprotein for rat α4β2) were incubated in PBS (50 μL and 100 μLrespectively) in the presence of competitor compound (0.01 nM to 100 μM)and 5 nM [³H]nicotine for 2-3 hours on ice. Incubation was terminated byrapid filtration on a multi-manifold tissue harvester (Brandel,Gaithersburg, Md.) using GF/B filters presoaked in 0.33%polyethyleneimine (w/v) to reduce non-specific binding. Tissue wasrinsed 3 times in PBS, pH 7.4. Scintillation fluid was added to filterscontaining the washed tissue and allowed to equilibrate. Filters werethen counted to determine radioactivity bound to the membranes by liquidscintillation counting (22000A Tri-Carb LSC, Packard Instruments, 50%efficiency or Wallac Trilux 1450 MicroBeta, 40% efficiency, PerkinElmer).

Data were expressed as disintegrations per minute (DPMs). Within eachassay, each point had 2-3 replicates. The replicates for each point wereaveraged and plotted against the log of the drug concentration. IC₅₀,which is the concentration of the compound that produces 50% inhibitionof binding, was determined by least squares non-linear regression. Kivalues were calculated using the Cheng-Pruss of equation (1973):Ki=IC₅₀/(1+N/Kd)where N is the concentration of [³H]nicotine and Kd is the affinity ofnicotine (3 nM, determined in a separate experiment).α7 NNR Subtype

Rats (female, Sprague-Dawley), weighing 150-250 g, were maintained on a12 h light/dark cycle and were allowed free access to water and foodsupplied by PMI Nutrition International, Inc. Animals were anesthetizedwith 70% CO₂, and then decapitated. Brains were removed and placed on anice-cold platform. The hippocampus was removed and placed in 10 volumes(weight:volume) of ice-cold preparative buffer (137 mM NaCl, 10.7 mMKCI, 5.8 mM KH₂PO₄, 8 mM Na₂HPO₄, 20 mM HEPES (free acid), 5 mMiodoacetamide, 1.6 mM EDTA, pH 7.4); PMSF, dissolved in methanol to afinal concentration of 100 μM, was added and the tissue suspension washomogenized by Polytron. The homogenate was centrifuged at 18,000× g for20 min at 4° C. and the resulting pellet was re-suspended in 10 volumesof ice-cold water. After 60 min incubation on ice, a new pellet wascollected by centrifugation at 18,000× g for 20 min at 4° C. The finalpellet was re-suspended in 10 volumes of buffer and stored at −20° C. Onthe day of the assay, tissue was thawed, centrifuged at 18,000× g for 20min, and then re-suspended in ice-cold PBS (Dulbecco's PhosphateBuffered Saline, 138 mM NaCl, 2.67 mM KCI, 1.47 mM KH₂PO₄, 8.1 mMNa₂HPO₄, 0.9 mM CaCl₂, 0.5 mM MgCl₂, Invitrogen/Gibco, pH 7.4) to afinal concentration of approximately 2 mg protein/mL. Protein wasdetermined by the method of Lowry et al., J. Biol. Chem. 193: 265(1951), using bovine serum albumin as the standard.

The binding of [³H]MLA was measured using a modification of the methodsof Davies et al., Neuropharmacol. 38: 679 (1999). [³H]MLA (SpecificActivity=25-35 Cl/mmol) was obtained from Tocris. The binding of [³H]MLAwas determined using a 2 h incubation at 21° C. Incubations wereconcluded in 48-well micro-titre plates and contained about 200 μg ofprotein per well in a final incubation volume of 300 μL. The incubationbuffer was PBS and the final concentration of [³H]MLA was 5 nM. Thebinding reaction was terminated by filtration of the protein containingbound ligand onto glass fiber filters (GF/B, Brandel) using a BrandelTissue Harvester at room temperature. Filters were soaked in de-ionizedwater containing 0.33% polyethyleneimine to reduce non-specific binding.Each filter was washed with PBS (3×1 mL) at room temperature.Non-specific binding was determined by inclusion of 50 μMnon-radioactive MLA in selected wells.

The inhibition of [³H]MLA binding by test compounds was determined byinduding seven different concentrations of the test compound in selectedwells. Each concentration was replicated in triplicate. IC₅₀ values wereestimated as the concentration of compound that inhibited 50 percent ofspecific [³H]MLA binding. Inhibition constants, (Ki values), reported innM, were calculated from the IC₅₀ values using the method of Cheng etal., Biochem. Pharmacol. 22: 3099-3108 (1973).

Selectivity vs. Peripheral nAChRs

Interaction at the Human Muscle nAChR Subtype

Activation of muscle-type nAChRs was established on the human clonalline TE671/RD, which is derived from an embryonal rhabdomyosarcoma(Stratton et al., Carcinogen 10: 899 (1989)). These cells expressreceptors that have pharmacological (Lukas, J. Pharmacol. Exp. Ther.251: 175 (1989)), electrophysiological (Oswald et al., Neurosci. Lett.96: 207 (1989)), and molecular biological profiles (Luther et al., J.Neurosci. 9: 1082 (1989)) similar to the muscle-type nAChR.

TE671/RD cells were maintained in proliferative growth phase accordingto routine protocols (Bencherif et al., Mol. Cell. Neurosci. 2: 52(1991) and Bencherif et al., J. Pharmacol. Exp. Ther. 257: 948 (1991)).Cells were cultured in Dulbecco's modified Eagle's medium (Gibco/BRL)with 10% horse serum (Gibco/BRL), 5% fetal bovine serum (HyClone, LoganUtah), 1 mM sodium pyruvate, 4 mM L-Glutamine, and 50,000 unitspenicillin-streptomycin (Irvine Scientific). When cells were 80%confluent, they were plated to 12 well polystyrene plates (Costar).Experiments were conducted when the cells reached 100% confluency.

Nicotinic acetylcholine receptor (nAChR) function was assayed using⁸⁶Rb⁺ efflux according to the method described by Lukas et al., Anal.Biochem. 175: 212 (1988). On the day of the experiment, growth media wasgently removed from the well and growth media containing ⁸⁶Rubidiumchloride (10⁵ μCi/mL) was added to each well. Cells were incubated at37° C. for a minimum of 3 h. After the loading period, excess ⁸⁶Rb⁺ wasremoved and the cells were washed twice with label-free Dulbecco'sphosphate buffered saline (138 mM NaCl, 2.67 mM KCI, 1.47 mM KH₂PO₄, 8.1mM Na₂HPO₄, 0.9 mM CaCl₂, 0.5 mM MgCl₂, Invitrogen/Gibco, pH. 7.4),taking care not to disturb the cells. Next, cells were exposed to either100 μM of test compound, 100 μM of L-nicotine (Acros Organics) or bufferalone for 4 min. Following the exposure period, the supernatantcontaining the released ^(ξ)Rb⁺ was removed and transferred toscintillation vials. Scintillation fluid was added and releasedradioactivity was measured by liquid scintillation counting.

Within each assay, each point had 2 replicates, which were averaged. Theamount of ⁸⁶Rb⁺ release was compared to both a positive control (100 μML-nicotine) and a negative control (buffer alone) to determine thepercent release relative to that of L-nicotine.

When appropriate, dose-response curves of test compound were determined.The maximal activation for individual compounds (Emax) was determined asa percentage of the maximal activation induced by L-nicotine. Thecompound concentration resulting in half maximal activation (EC₅₀) ofspecific ion flux was also determined.

Interaction at the Rat Ganglionic nAChR Subtype

Activation of rat ganglion nAChRs was established on thepheochromocytoma clonal line PC12, which is a continuous clonal cellline of neural crest origin, derived from a tumor of the rat adrenalmedulla. These cells express ganglion-like nAChR s (see Whiting et al.,Nature 327: 515 (1987); Lukas, J. Pharmacol. Exp. Ther. 251: 175 (1989);Whiting et al., Mol. Brain Res. 10: 61 (1990)).

Rat PC12 cells were maintained in proliferative growth phase accordingto routine protocols (Bencherif et al., Mol. Cell. Neurosci. 2: 52(1991) and Bencherif et al., J. Pharmacol. Exp. Ther. 257: 946 (1991)).Cells were cultured in Dulbecco's modified Eagle's medium (Gibco/BRL)with 10% horse serum (Gibco/BRL), 5% fetal bovine serum (HyClone, LoganUtah), 1 mM sodium pyruvate, 4 mM L-Glutamine, and 50,000 unitpenicillin-streptomycin (Irvine Scientific). When cells were 80%confluent they were plated to 12 well Nunc plates (Nunclon) and coatedwith 0.03% poly-L-lysine (Sigma, dissolved in 100 mM boric acid).Experiments were conducted when the cells reached 80% confluency.

Nicotinic acetylcholine receptor (nAChR) function was assayed using⁸⁶Rb⁺ efflux according to a method described by Lukas et al., Anal.Biochem. 175: 212 (1988). On the day of the experiment, growth media wasgently removed from the well and growth media containing ⁸⁶Rubidiumchloride (10⁶ μCi/mL) was added to each well. Cells were incubated at37° C. for a minimum of 3 h. After the loading period, excess ⁸⁶Rb⁺ wasremoved and the cells were washed twice with label-free Dulbecco'sphosphate buffered saline (138 mM NaCl, 2.67 mM KCI, 1.47 mM KH₂PO₄, 8.1mM Na₂HPO₄, 0.9 mM CaCl₂, 0.5 mM MgCl₂, Invitrogen/Gibco, pH. 7.4),taking care not to disturb the cells. Next, cells were exposed to either100 μM of test compound, 100 μM of nicotine or buffer alone for 4 min.Following the exposure period, the supernatant containing the released⁸⁶Rb⁺ was removed and transferred to scintillation vials. Scintillationfluid was added and released radioactivity was measured by liquidscintillation counting

Within each assay, each point had 2 replicates, which were averaged. Theamount of ⁸⁶Rb⁺ release was compared to both a positive control (100 μMnicotine) and a negative control (buffer alone) to determine the percentrelease relative to that of L-nicotine.

When appropriate, dose-response curves of test compound were determined.The maximal activation for individual compounds (Emax) was determined asa percentage of the maximal activation induced by L-nicotine. Thecompound concentration resulting in half maximal activation (EC₅₀) ofspecific ion flux was also determined.

Novel Object Recognition

Memory was assessed by using the three-trial novel object recognitiontest. On the first day (exploratory trial), rats were allowed to explorean open arena (44.5×44.5×30.5 cm) for 6 min. On the second day(acquisition trial), rats were allowed to explore the same arena in thepresence of two identical objects (both object A) for 3 minutes. On thethird day (retention or recall trial), performance was evaluated byallowing the same animal to re-explore the arena for 3 minutes in thepresence of two different objects: the familiar object A and a novelobject B. An inter-trial interval of 24 hours was imposed between thethree NOR trials. Recognition memory was assessed by comparing the timespent exploring a novel (object B) versus a familiar (object A) objectduring the recall trial. Recognition index was assessed for each animaland expressed as a ratio ((time B/time A+time B)×100).

Summary of Biological Data

In vitro Pharmacology

A summary of the in vitro primary pharmacology data for(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide,or a pharmaceutically acceptable salt thereof, is presented in Table 1and discussed in detail below.

Primary pharmacology and selectivity:(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideinhibited the binding of [³H]methyllycaconitine (MLA) to rat native α7receptors in rat hippocampal membranes with a K_(i) of 100 nM.

(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideinhibited the binding of [³H]-nicotine to human recombinant α4β2nicotinic receptors with a K_(i) of 1470 nM and [³H]epibatidine to ratnative α4β2 receptors with a K_(i) of 4120 nM.(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidealso displayed reduced affinity for human native ganglion-type nicotinicreceptors (likely α3β4), inhibiting the binding of [³H]epibatidine toreceptors in SH-SY5Y membranes with a K_(i) of 48 μM, and reducedaffinity for human native muscle-type nicotinic receptors (likelyα1β1γδ), inhibiting the binding of [³H]epibatidine to receptors inTE-671 membranes with a K_(i) of 136 μM.(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideinhibited the binding of [³H]epibatidine to the human recombinant α4β4nicotinic receptors in SH-EP1 membranes with a K_(i) of 19 μM.

TABLE 1 Summary of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide in vitro pharmacology Target affinity andactivation Rat hippocampus (α7), K_(i) 0.1 μM Rat cortex binding K_(i)4.12 μM Human recombinant (SH-EP 1) α4β2 binding K_(i) 1.47 μM Humanganglionic (SH-SY5Y), K_(i) 48 μM Human (TE671/RD) muscle, K_(i) 136 μMHuman recombinant (SH-EP1) α4β4, K_(i) 19 μMIn vivo Pharmacology

A summary of the in vivo pharmacology data for(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide,or a pharmaceutically acceptable salt thereof, is presented in Table 2and discussed in detail below.

TABLE 2 Summary of NOR results for (2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide, or apharmaceutically acceptable salt thereof Novel Object Recognition Model(NOR) Result Minimum Effective Dose (MED) MED = 0.084 μmol/kg Durationof Effect Duration 6 h (@ 0.1 mg/kg) Duration 18 h (@ 0.3 mg/kg)

(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicydo[2.2.2]oct-3-yl)-3,5-difluorobenzamideimproved long-term visual episodic/declarative memory as assessed bynovel object recognition (NOR) task following oral dosing in normalrats. The results of these studies are presented in FIG. 1. Therecognition index of the vehicle-treated group 24 h after theacquisition trial was 54±1% demonstrating the inability of this group torecognize the familiar object after this delay. By contrast, animalstreated with(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideexhibited recognition indexes of 70±4% at the 0.84 μmol/kg dose leveland 74±3% and the 0.28 μmol/kg dose level.

In a follow-up NOR study (FIG. 2), the minimum effect dose (MED) levelfor(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidewas determined to be 0.084 μmol/kg suggesting that the rats are able torecognize the familiar object at all doses levels tested. In the “recallonly” session; a subset of animals were orally dosed with water on day 1(i.e., exploratory session) and day 2 (i.e., acquisition session) andthen orally dosed with 0.28 μmol/kg(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideon day 3 (i.e., recall session). Even following a single oraladministration,(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidedemonstrated pro-cognitive effects at this dose level.(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideexhibited recognition indexes significantly above controls, indicatingrecognition of the familiar object following acute dosing. The dashedline at 65% denotes our criteria for biological cognitive enhancingactivity. *P<0.05.

(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidewas evaluated for its duration of effect in the NOR task in normal rats.The results of these studies are presented in FIG. 3. The recognitionindex of the vehicle-treated group at 0.5 h and 24 h following dosing onthe recall trial was 51±1% and 53±4%, respectively, demonstrating theinability of this group to recognize the familiar object after thisdelay. By contrast, animals treated with(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(0.28 μmol/kg: oral) exhibited recognition indexes of 68±4% at 0.5 h,71±2% at 2 h and 62±2% at 6 h suggesting that rats are able to recognizethe familiar object for up to 6 h after dosing.

Furthermore, animals treated with(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(0.84 μmol/kg: oral) exhibited recognition indexes of 59±2% at 0.5 h,63±2% at 2 h, 68±3% at 6 h, and 68±3% at 18 h suggesting that rats areable to recognize the familiar object for up to 18 h after dosing atthis dose level (FIG. 4). The dashed line at 65% denotes our criteriafor biological cognitive enhancing activity (*P<0.05).

Electrophyslology

Compound A,(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide,and Compound B,(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-4-fluorobenzamideare both partial agonist at the α7 NNR. However dramatic differencesexist between the two compounds in their ability to induce so-calledhump currents. Hump currents are defined as the tail current observedduring co-application with endogenous ACh following agonist removal. Asdemonstrated herein, Compound A provides an improved profile and agreater potential to modulate α7 function in conditions, such aspsychotic disorders, where this neurotransmission is compromised.

The dose-response of Compounds A and B with α7 nicotinic ACh receptorswas analyzed. Both Compound B and Compound A are partial agonists at α7nicotinic receptors (EC₅₀=664 nM, 1.6 μM and E_(MAX)=46.6%, 54.4%). Asshown in FIG. 5, both EC₅₀ and E_(MAX) are comparable between theseligands.

Co-application of compounds with ACh, however, revealed substantialdifferences between these two ligands, as illustrated in FIG. 6.Compound B inhibited current produced by ACh, presumably due tocompetitive inhibition, whereas Compound A enhanced ACh-induced current.One hypothesis for this enhancement is Compound A's ability fororthosteric modulation.

Additionally, substantial differences were found when ACh was co-appliedwith nanomolar concentrations of Compound B or Compound A, as shown inFIGS. 7 and 8.

FIG. 7A represents an experimental design of loading Dynaflow chip tomeasure interaction of the ligand (Compound A, 200 nM) withacetylcholine (100 μM) regarding activation of nicotinic α7 receptor.The channels were prepared as follows: Control solution (channel #2),application ligand itself (channel #3), application of acetylcholineitself (channel #1), and application of mixture of acetylcholine andligand (channel #4).

FIG. 7B shows four representative current curves obtained with differentapplication sequences:

Curve 1, FIG. 7B: The bar above the curve indicates time of AChapplication. The curve represents current induced by a one secondapplication of 70 μM ACh. The curve illustrates the result from movingthe cell from channel #2 to channel #1 for a 1 second application of AChand back to channel 2 (washout). The application of ACh produced robustactivation of current with fast recovery after washout.

Curve 4, FIG. 7B: Curve 4 represents a repetition of Curve 1 at the endof measurements after application of the ligand and ACh/ligand mixture(recovery).

Curve 2, FIG. 7B: The down/up arrows indicates time of application.Curve 2 represents a 5 second application of 200 nM of Compound A. Thecurve illustrates the result of moving the cell from channel #2 tochannel #3 for 5 seconds. Compound A in concentration of 200 nM alone donot produce robust macro currents.

Curve 3, FIG. 7B: Curve 3 represents the interaction of the applicationof ACh and Compound A. Curve 3 illustrates the results from moving thecell from channel #2 to #3 (2 sec), to #4 (1 sec) and back to #3 (2 sec)and back to #2 (washout). A profound “hump” current is created due toapplication of Compound A following application of ACh. This current wasnot a result of ACh, as seen when comparing Curves 1 and 4, or CompoundA, as seen in Curve 2, activation of α7 receptors alone. Rather Curve 3illustrates an example of interaction of application of both ACh andCompound A.

FIG. 7C represents an average (n=4) of absolute values of hump currentsobtained with different concentrations of Compound A (100-500 nM range).We observed a concentration dependent increase of current (EC₅₀=120 nM)with E_(MAX) at approximately 500 nM.

Similarly, FIGS. 8A, 8B, and 8C represent the results obtained forCompound B. Upon comparing FIGS. 7A-C and with FIGS. 8A-C, substantialdifferences may be noted when ACh was co-applied with Compound A ascompared to Compound B. Compound A enhances ACh-induced current.

Formalin Test

One of the most clinically predictive screening models of acute pain isthe formalin test in mice (LeBars et al., 2001). In this paradigm,originally described by Dubuisson and Dennis (1977), a diluted solutionof formalin is injected into the plantar surface of a subject's (rat ormouse) rear paw and nociceptive behavior is measured; for instance,licking and biting of the injected paw. Two phases of the response areobserved. First an early phase, starting immediately after injection andlasting 5-10 minutes, followed by a late phase that can last from 15-60minutes after injection. Nociceptive response is attributed to directchemical stimulation in the early phase and inflammation/persistent painin the later phase (Dubuisson and Dennis, 1977). The response in thelate phase also depends on changes in processing of information in thespinal cord due to the afferent barrage during the early phase (Coderreet al., 1990). An advantage of the test is that two different types ofstimuli are employed in the same assay to study the possibility ofvarying analgesic effects of a drug in the two phases of the test(Tyolsen and Hole, 1997).

Subjects (adult male CD-1 mice (Charles River, Raleigh, N.C.) weighingapproximately 20-25 grams) were removed from their home cage andweighed, then placed in a clear Plexiglas™ observation box for anacclimation period of 20-30 minutes. Mice were then removed from theobservation chambers and(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(as the hydrochloride salt in 0.9% saline) (1, 3 or 10 mg/kg s.c.(calculated with respect to the free base)), morphine (5 mg/kg; s.c.) or0.9% saline vehicle was administered subcutaneously in a volume of 1ml/kg. Mice were then returned to the chamber for the predeterminedpretreatment time of 30 minutes for(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideand morphine.

After the test compound pretreatment time, the animals were injectedwith formalin solution (2.5% derived as a 1:4 dilution of 10% phosphatebuffered formalin solution (Sigma):distilled water). The subject'sassigned paw was grasped gently and formalin solution was injected intothe paw intra-dermally in the middle on the dorsal side. Once injected,the subject was immediately returned to its observation chamber and atimer was started to mark the beginning of Phase I. Each subject wasvideotaped for the entire 40-minute session. When scoring the tapes,each subject was observed for 1 min at 5-min intervals over a 40-minutesession. The time spent licking during that 1 min interval was recorded,and the presence or absence of paw favoring was noted.

For data analyses, phase I of the test was defined as 0 to 5 minutesafter formalin injection, and phase II was defined as 20 to 40 minutesafter formalin injection. The time spent licking during the 1 minuteintervals during those time frames was recorded and graphed asmean±S.E.M. For comparisons across treatment groups, 1-way analyses ofvariance (ANOVAs) were performed for each phase of the session withtreatment as the dependent variable. Post-hoc analyses were performedwhen appropriate to determine specific group differences.

The results demonstrate that although there was no statisticallysignificant dose of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidein reducing time spent licking in phase I, nevertheless, 10 mg/kg(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidewas significant in reducing the time spent licking the paw in phase IIof the formalin test (P<0.05). The positive control morphine (5 mg/kg;s.c.) was efficacious in both phases of the test. These data indicatethat(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidehas analgesic potential with respect to chemically-inducedinflammatory/persistent pain.

Subsequent analysis of the original videotapes wherein each animal wasscored across the entire time period for phase I (0-5 min afterformalin) and phase II (20-40 min) revealed a similar trend for thedata, but failed to achieve statistical significance for the effect of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideon the reduction of time spent licking the affected paw in either phaseI or phase II.

Reference is made to: Coderre T J, Vaccarino A L, Melzack R (1990),Central nervous system plasticity in the tonic pain response tosubcutaneous formalin injection, Brain Res. 535:155-158; Dubuisson D andDennis S G (1977), The formalin test: A quantitative study of theanalgesic effects of morphine, meperidine, and brain stem stimulation inrats and cats, Pain 4: 161-174; Malmberg A B and Bannon A W (2002), Unit8.9: Models of nociception: hot-plate, tail-flick, and formalin tests inrodents, Current Protocols in Neuroscience; and Tjolsen A and Hole K(1997), Animal models of analgesia, In: Handbook of ExperimentalPharmacology Volume 130: The Pharmacology of Pain (Eds. A. Dickenson andJ.-M. Besson), Springer Verlag, New York pp. 1-20. Complete Freund'sAdjuvant (CFA)-Induced Thermal Hyperalgesia

Injection of complete Freund's adjuvant (CFA) in rats is commonly usedto evaluate compounds with potential for use as drugs in treatment ofmono-arthritis (osteo-arthritis) and other inflammatory conditions.Signs of hyperalgesia develop within 24 h (Schaible and Grubb, 1993).

(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidewas evaluated for possible analgesia effect in the CFA-induced thermalhyperalgesia test in rats using methods similar to those described byWalker and colleagues (2003). Briefly, adult male Sprague-Dawley rats(BioLasco, Taiwan) weighing 180±20 g on receipt were randomly assignedto treatment groups of n=8 per group. Animals each received a subplantar injection (0.1 mL) of CFA (DIFCO, 264010; 0.1% solution) to theright hind paw at 24 h prior to experimental testing. Thermalhyperalgesia was tested using a Paw/Tail stimulator analgesia meter(IITC Model-336G, IITC, USA) with a thermally regulated glass floor setat 30° C. A subject was placed within a plastic box atop an elevatedglass floor and a light beam located under the glass floor was directedat the plantar surface of the right hind paw. The time required for theanimal to withdraw the paw from the thermal stimulus was automaticallyrecorded. The intensity of the light was adjusted to evoke an averagegroup baseline latency from 12-14 seconds (pre-CFA) and a cut-offlatency of 20 seconds was imposed. The latency for paw withdrawal wasobtained for each rat and defined as the heat pain threshold.

Twenty-four hours after CFA injection, subjects were pre-selected (forclear presence of thermal hyperalgesia) for experimentation only if thelatency to withdrawal was less than 75% of the baseline. Test substance,morphine and vehicle were administered by subcutaneous (s.c.) injectionat time 0. The post-treatment level of thermal hyperalgesia was thenmeasured at 60 minutes post-treatment. One-way ANOVA followed byDunnett's test was applied for comparison between test substance treatedgroups and vehicle control group. Activity was considered significant atthe P<0.05 level.

Overall, subcutaneous (s.c.) administration of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicydo[2.2.2]oct-3-yl)-3,5-difluorobenzamideat 0.1, 1 or 10 mg/kg was not associated with any significant analgesiceffect at 1 hour post-dose on CFA-induced thermal hyperalgesia in ratscompared with the vehicle (0.9% saline) control group. In contrast, theconcurrently run reference standard morphine (3 mg/kg s.c.) producedsignificant analgesic activity at 1 hour after dosing. See FIG. 13.Reference is made to: Schaible H-G and Grubb B D (1993), Afferent andspinal mechanisms of joint pain, Pain 55: 5-54; and Walker K M, Urban L,Medhurst S J, Patel S, Panesar M, Fox A J and Mcintyre P (2003), The VR1antagonist capsazepine reverses mechanical hyperalgesia in models ofinflammatory and neuropathic pain, JPET 304: 56-62.

Streptozotocin (STZ)—Induced Diabetic Neuropathy (As Evidenced byAllodynia)

Peripheral neuropathy, a major complication of diabetes, often resultsin spontaneous pain or the perception of pain from contact with anormally non-noxious stimulus. Such neuropathic pain is experienced by20-24% of diabetic patients, or approximately 30 million peopleworldwide (Schmader, 2002). The streptozotocin (STZ)-induced diabetesmodel in rats provides a means to evaluate the efficacy of testcompounds that offer therapeutic potential for peripheral neuropathy andto understand their putative mechanism of action. In this model, asingle injection of STZ, an antibiotic that mimics clinical diabetes bycausing irreversible damage to the pancreatic β and α-cells leads tochronic hyperglycemia, nerve dysfunction and pain sensitivity. Thecurrent study utilizes the STZ rat model of diabetic neuropathy toinvestigate the effects of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideon mechanical allodynia, an assessment of pain.

Diabetes was induced by a 0.5 ml injection of streptozotocin (60 mg/kg)dissolved in citrate buffer (pH=6) into the tail vein of each rat. Thedevelopment of diabetes was confirmed by measuring the blood glucoselevels (BGL) of all animals on study day 3 (BGL>300 mg/dL). BGL wasmeasured again on study day 14 and only the animals that showed tactileallodynia were tested again for their BGL on study day 21. BGL wasmeasured on study day 16 for animals that did not show tactile allodyniaon study day 14. These animals were tested again for their BGL at studyday 23.

(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(0.1, 1 or 10 mg/kg p.o.) was administered as a solution of thehydrochloride salt in water once daily starting on study day 14 or studyday 16 and continuing through study day 21 or 23, respectively. Thecontrol article gabapentin (in 0.9% saline, 150 mg/kg i.p.) was onlyadministered on allodynia test days. Test item, vehicle or controlarticle administration was based on evaluation of allodynia on study day14. If allodynia was not present on study day 14, the animal wasevaluated again at study day 16. Pain response was measured either onstudy days 14 and 21 or 16 and 23, 30 minutes after Test itemadministration.

For the allodynia assessments, Von Frey filaments were used according tothe methods of Chaplan and colleagues (1994). Briefly, the rats wereplaced in an enclosure and positioned on a metal mesh surface, butallowed to move freely. The rats' cabins were covered with redcellophane to diminish environmental distributions. The test began aftercessation of exploratory behavior.

Rodents exhibit a paw withdrawal reflex when its paw is unexpectedlytouched. When the tip of a Von Frey fiber of given length and diameterwas pressed against the skin at right angles, the force of applicationincreases as long as the researcher continued to advance the probe untilthe fiber bent. After the fiber bent, the probe was advanced, causingthe fiber to bend more, but without additional force being applied. Theanimal would indicate sensation by pulling back its paw. In the absenceof a paw withdrawal response to the initially selected filament, astronger stimulus was presented; in the event of paw withdrawal, thenext weaker stimulus was chosen. In this fashion, the resulting patternof positive and negative responses was used to determine the pawwithdrawal threshold.

The set of Von Frey monofilaments provide an approximate logarithmicscale of actual force and a linear scale of perceived intensity. Belowis a table showing the force (g) and its corresponding size ofmonofilaments.

Size 1.65 2.36 2.44 2.83 3.22 3.61 3.84 4.08 4.17 4.31 4.56 4.74 4.935.07 5.18 5.46 5.88 6.10 6.45 6.65 Force 0.008 0.02 0.04 0.07 0.16 0.400.60 1.00 1.40 2.00 4.00 6.00 8.00 10 15 26 60 100 180 300 (g)

All normally distributed data are presented as means±SEM, as well as theanimals' individual values followed by a student T-test (Software:Microsoft® Excel). A p value <0.05 is considered to represent asignificant difference. Due to the non-normal distribution of theallodynia data, descriptions of those data are provided as both mean(±SEM) and median values in order to represent their imprecise natureand skewed distribution.

The Von Frey data are presented as the minimum force (g) needed towithdraw each hind leg. A decrease in pain threshold was recorded 14/16days post STZ injection. This decrease was expressed as an increase inthe animal's sensitivity to the Von Frey filaments. The average andgroup median withdrawal force of the vehicle treated animals at baselinebefore STZ injection was 57.57±2.43 (group median=60 g). On study days14/16, the median paw withdrawal force was significantly lower(20.5-22.14±2.36 g; <0.01 vs. baseline; median=20.5 g) indicatingtactile allodynia prior to(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidetreatment. At study termination (study days 21/23), tactile allodyniawas still observed post treatment (20.46±3.31 g; p<0.01 vs. baseline;median=8 g).

Overall, Treatment with 1 mg/kg(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideinhibited allodynia 30 minutes after its administration on study days14/16 as compared to pretreatment (p<0.01) or to the Vehicle control(p<0.05). Treatment with(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideat a dose of 10 mg/kg inhibited allodynia 30 minutes after itsadministration on study days 14/16 as compared to pretreatment(p=0.012). Treatment with(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideat a dose of 1 mg/kg inhibited allodynia 30 minutes after theiradministration on study days 21/23 as compared to pretreatment(p=0.012). Treatment with(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideat a dose of 10 mg/kg (Group 11M) inhibited allodynia 30 minutes afterits administration on study days 21/23 as compared to the Vehiclecontrol (p<0.05). Treatment with the positive control, gabapentin,reversed the tactile allodynia significantly in all treatment days ascompared to pretreatment (study days 14/16 and 21/23; p<0.01) or ascompared to the Vehicle control (study days 21/23; p<0.01). At studytermination (study day 21/23), insulin levels in the serum wereanalyzed. No significant differences in insulin levels were observed.(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideat all doses was administered every day starting on study day 14 or 16through study day 21 or 23, respectively. The pain test was performedprior to Test Article injection (pre-TI injection) and 30 minutes afterTest Article administration (post-TI injection). The positive control,gabapentin, was administered 2 hours before pain testing on study days14 or 16 and 21 or 23. Treatment with the positive control, gabapentin,reversed the tactile allodynia significantly in all treatment days ascompared to pretreatment and to the vehicle: 22.77±3.77 g (median=15 g)vs. 45.62±4.24 g (median=60 g) in pre and post treatment, respectively,on study days 14/16, p<0.01; 28.23±4.91 g (median=20.5 g) vs. 50.88±4.12g (median=60 g) in pre and post treatment, respectively, on study days21/23, p<0.01; 45.62±4.24 g (median=60 g) vs. 26.61±4.41 g (median=15 g)in the vehicle group on study days 14/16, p<0.01; 50.88±4.12 g(median=60 g) vs. 20.46±3.31 g (median=10 g) in the vehicle group onstudy days 21/23, p<0.01.

Immediately after the Von Frey testing on the termination days, bloodwas collected. At the end of the study, the animals were euthanized withketamine/ylazine solution (IP). Approximately 0.5-0.7 ml of blood wascollected via cardiac puncture in tubes containing the anti-coagulant(K3 EDTA). The blood samples were kept chilled on ice and centrifugedwithin 30 minutes of collection. To obtain plasma, blood was centrifugedfor 10 minutes at 3000 rpm. Plasma was transferred into labeled tubesand stored upright and frozen at approximately −20° C. until shipment.Each sample was labeled with the compound number and animal number.

All animals gained weight during the study. There were no significantdifferences in body weight gain between the groups.

The mean blood glucose levels increased in all animals. Baseline was108.86±1.03 mg/dl and increased to 390.99±6.47 mg/dl on study day 3. Nostatistical differences were found between groups. High glucose levelswere also measured on study days 14/16 and 21/23 based on the resultsfor allodynia at study day 14. At the end of the study (study day 21 and23) the mean blood glucose level was 403.86±8.45 mg/dl.

At study termination, insulin levels in serum were analyzed. The insulinlevel in the Vehicle control at study termination was 0.79±0.41 μg/l. Nosignificant differences in insulin levels between treatments wereobserved.

The results of Von Frey assessment indicate that(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideis effective in reducing diabetic neuropathy pain at doses of 1 mg/kgand 10 mg/kg compared to the Vehicle treated group. See FIG. 14.

Reference is made to: Chaplan S R, Bach F W, Pogrel J W , Chung J M,Yaksh T L (1994), Quantitative assessment of tactile allodynia in therat paw, J. Neurosci. Methods 53: 55-63; Schumader K E (2002),Epidemiology and impact on quality of life of postherpetic neuralgia andpainful diabetic neuropathy, Clinical Journal of Pain 18: 350-354; andSommer C (2003). Painful neuropathies, Curr. Opin. Neurol. 16: 623-628.

Murine Model of Type 2 Diabetes Mellitus

The db/db mouse, a well established model of type 2 diabetes mellitus,is a leptin-deficient mutant that expresses an obese phenotype and alsocommonly expresses metabolic symptoms including hyperglycemia,hyperlipidemia and hyperinsulinemia (Halaas et al., 1995 and Lee et al.,1996). This experimental animal model of diabetes was employed in astudy designed to determine the effects of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideon body mass and several additional metabolic parameters.

In this study,(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidewas repeatedly administered orally (via gavage) at 1.0 mg/kg once dailystarting from the age of approximately 3 weeks and continuing throughoutthe 7-week study. Nondiabetic heterozygote littermate mice (db/+;designated “Db”) were used as controls. Body weight and food intake weredetermined twice weekly. The α7 antagonist methyllycaconitine (MLA) wasalso given concurrently via gavage at 3 mg/kg daily to selected cohortsof db/db (designated “db”) or db/+ mice. At the end of the 7-week dosingregimen, total growth rates (overall body weight gain) and average dailyfood intake were calculated. In addition, glucose levels were assessedin mice fasted overnight. Furthermore, blood sample analytes from micefasted overnight were collected for measurements of tumor necrosisfactor-a (TNF-α), triglycerides and glycosylated hemoglobin (HbA1c). Alldata are expressed as mean±SEM. For each parameter investigated,differences among all groups were compared by one-way ANOVA withpost-hoc Neuman-Keuls multiple comparison test.

Overall, daily administration over the course of 7 weeks of(2S,3R)-N-2-((3-pyridinyl)methy)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideto obese db/db mice resulted in a significant decrease in all parametersmeasured compared with control obese db/db mice treated with vehicle.With respect to total body weight gain, average daily food consumption,glycosylated HbA1c levels and plasma concentration of TNF-α,co-administration of MLA attenuated the effect. Although attenuation ofplasma glucose and triglycerides was not significantly attenuated byco-administration of MLA with(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide,there was a trend toward that reversal.

As illustrated in FIG. 15, At the end of seven weeks of treatment,between ages 3 and 10 weeks, total body weight gain in the vehiclecontrol-treated obese group (“db”) was significantly greater than thatof lean vehicle control animals (“Db”). By comparison, weight gain wassignificantly lower in the(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide-treatedobese (“db-Test Article”) mice. Notably, animals that wereco-administered MLA with(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidefailed to show the reduced weight gain exhibited by the obese ratsadministered(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidealone.

As shown in FIG. 16, The daily food intake in vehicle control obesegroup (“db”) was significantly greater than that of lean vehiclecontrols (“Db”). Average food consumption was significantly lower in theTC-(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide-treatedobese mice (“db-Test Article”) than in the obese controls. The foodconsumption of the lean mice was unaffected by(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide(“Db-Test Article”). Animals that were co-administered MLA with(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidefailed to show the reduced daily average food consumption exhibited bythe obese rats administered(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidealone.

As shown in FIG. 17,(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidesignificantly inhibited fasting plasma glucose levels in obese mice(“db-Test Article”). However, this effect was not reversed byco-administration with MLA.

As shown in FIG. 18,(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidesignificantly inhibited glycosylated HbA1c levels in obese nice(“db-Test Article”). The reduction in glycosylated HbA1c by(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidewas attenuated by co-administration of MLA.

As shown in FIG. 19,(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicydo[2.2.2]oct-3-yl)-3,5-difluorobenzamidesignificantly reduced the pro-inflammatory cytokine TNF alpha in obesemice (“db-Test Article”). These effects were inhibited byco-administration of the alpha7 antagonist MLA.

As shown in FIG. 20,(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideresulted in significantly lower triglyceride levels in obese nice(“db-Test Article”) compared with vehicle-treated controls (“db”). Thereduction in triglycerides by(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicydo[2.2.2]oct-3-yl)-3,5-difluorobenzamidewas not attenuated by co-administration of MLA.

Pulmonary, Airway Hyperresponsiveness, Penh Measurement

Using the method of Hamelmann et al,(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicydo[2.2.2]oct-3-yl)-3,5-difluorobenzamidewas evaluated for possible inhibition of airway hyper-responsiveness inmice. Breifly, ovalbumin (OVA)-sensitized animals, 12 animals per group,were challenged by nasal inhalation with aerosolized 5% OVA for 25 minon days 21, 23, and 25. The mice were treated with vehicle or(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamidesubcutaneously (s.c.) twice daily from day 21 to day 26 or once dailyintratracheally (i.t.), preceding ovalbumin aerosol challenge by 30 minon days 21, 23 and 25 as well as methacholine challenge orbronchoalveolar lavage fluids (BALF) harvest on day 26. Dexamethasone,the reference standard, was administered at 3 mg/kg orally (p.o.) oncedaily 60 min before OVA challenge on day 21, 23 and 25 and 60 min beforemethacholine provocation or BALF harvest on day 26. Noninvasivemeasurements of airway responsiveness were performed by using whole bodyplethysmography, in which increases in enhanced pause (Penh) serve as anindex of airway obstruction. Responses to inhaled methacholine weremeasured and calculated as percentage of respective baseline values.Unpaired Student's t-test was used for comparison between the vehiclecontrol and the sham group; one-way ANOVA and Dunnett's post-hocanalyses were applied for comparison between the vehicle control andtreated groups. Statistical significance is considered at P<0.05.

FIG. 21 illustrates the effect of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideon % changes in Penh response to methacholine challenge inovalbumin-sensitized mice. The Penh response to methacholine (10 and 30mg/mL) was significantly augmented in OVA-sensitized animals compared tosham control. Dexamethasone at 3 mg/kg PO caused a significantinhibition of the methacholine (10 and 30 mg/mL)-induced increase inPenh values, both in absolute and % values compared to vehicle-treatedOVA animals, indicating efficacy against airway hyperresponsiveness.(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideat 0.1, 1, and 10 mg/kg bid s.c. caused significant inhibition of themethacholine-induced increase in Penh values; 10 mg/kg IT was alsoassociated with significant inhibition.

The effect of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideon white blood cell counts/differential cell counts and % white bloodcell count/differential cell counts in ovalbumin sensitized mice areillustrated in Figures Y and Z, respectively. A significant increase intotal WBC, neutrophils, lymphocytes, monocytes and eosinophils was notedin BALF in OVA-sensitized animals vs. sham control, which was inhibitedsignificantly by dexamethasone.(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideat 0.1 and 1 mg/kg SC, but not at 10 mg/kg SC, significantly reducedtotal WBC and eosinophils in BALF;(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideat 10 mg/kg SC reduced monocytes; lymphocytes were reduced at 0.1 and 1mg/kg SC as well as at 10 mg/kg IT.

These results demonstrate that multiple administrations of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideat 0.1, 1 and 10 mg/kg bid s.c. and at 10 mg/kg i.t. vs dexamethasoneaffords significant protection against airway hyper-responsiveness inOVA sensitized mouse model (as evidenced by reduced Penh response tomethacholine challenge using whole body plethysmography in mice) and isassociated with significant reduction in eosinophils and white bloodcells in BALF (which, however, lacks a consistent dose-responserelationship).

FIG. 21 illustrates the effect of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideon % changes in Penh response to methacholine challenge inovalbumin-sensitized mice.(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideand vehicle were administered subcutaneously bid or givenintratracheally qd for 6 consecutive days from day 21 to day 25 at 30min before OVA challenge and the last dosing was administrated at 30 minbefore MCh provocation on day 26. The Penh values were determined.One-way ANOVA followed by Dunnett's test was applied for comparisonbetween the OVA immunized vehicle and other treatment groups. *P<0.05vs. OVA-vehicle control.

FIG. 22 illustrates the effect of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideon white blood cell counts and differential cell counts in ovalbuminsensitized mice.(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideand vehicle were administered subcutaneously bid or were givenintratracheally qd for 6 consecutive days from day 21 to day 25 at 30minutes before OVA challenge and the last dosing was administrated at 30minutes before bronchoalveolar lavage fluid harvest on day 26. The totalwhite blood cell count and differential cell counts were determined.One-way ANOVA followed by Dunnett's test was applied for comparisonbetween the OVA immunized vehicle and other treatment groups. *P<0.05vs. OVA-vehicle control.

FIG. 23 illustrates the effect of(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamideon % white blood cell count and differential cell counts in ovalbuminsensitized mice.(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicydo[2.2.2]oct-3-yl)-3,5-difluorobenzamideand vehicle were administered subcutaneously bid or were givenintratracheally qd for 6 consecutive days from day 21 to day 25 at 30minutes before OVA challenge and the last dosing was administrated at 30minutes before bronchoalveolar lavage fluid harvest on day 26. The totalwhite blood cell count and differential cell counts were determined.One-way ANOVA followed by Dunnett's test was applied for comparisonbetween the OVA immunized vehicle and other treatment groups. *P<0.05vs. OVA-vehicle control.

Reference is made to: Hamelmann E, Schwarze J, Takeda K, Oshiba A,Larsen G L, Irvin C G, and Gelfand E W, Noninvasive measurement ofairway responsiveness in allergic mice using barometric plethysmography,Am J Respir Crit Care Med, 156:766-775,1997.

Test compounds for the experiments described herein were employed infree or salt form. Unless otherwise specified, the compound provided forin vivo testing was(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicydo[2.2.2]oct-3-yl)-3,5-difluorobenzamidehydrochloride, with dosage amounts given assuming the free base form.

The specific pharmacological responses observed may vary according toand depending on the particular active compound selected or whetherthere are present pharmaceutical carriers, as well as the type offormulation and mode of administration employed, and such expectedvariations or differences in the results are contemplated in accordancewith practice of the present invention.

Although specific embodiments of the present invention are hereinillustrated and described in detail, the invention is not limitedthereto. The above detailed descriptions are provided as exemplary ofthe present invention and should not be construed as constituting anylimitation of the invention. Modifications will be obvious to thoseskilled in the art, and all modifications that do not depart from thespirit of the invention are intended to be included with the scope ofthe appended claims.

That which is claimed is:
 1. A compound(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamide(Formula I) or a pharmaceutically acceptable salt thereof


2. The compound of claim 1, which is substantially free of one or moreof(2R,3S)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamide,(2R,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamide,and(2S,3S)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamide.3. The compound of claim 1, as an acid addition salt, wherein the acidis selected from hydrochloric acid, methanesulfonic acid, maleic acid,phosphoric acid, 1-hydroxy-2-naphthoic acid, malonic acid, L-tartaricacid, fumaric acid, citric acid, L-malic acid, R-mandelic acid,S-mandelic acid, succinic acid, 4-acetamidobenzoic acid, adipic acid,galactaric acid, di-p-toluoyl-D-tartaric acid, oxalic acid, D-glucuronicacid, 4-hydroxybenzoic acid, 4-methoxybenzoic acid,(1S)-(+)-10-camphorsulfonic acid, (1R,3S)-(+)-camphoric acid, andp-toluenesulfonic acid.
 4. The compound of claim 3, wherein the molarratio of acid to(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamideis 1:2 or 1:1.
 5. A compound selected from:(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamidemono-hydrochloride;(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamidemono-phosphate;(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamidemono-4-hydroxybenzoate; and(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamidehemi-4-hydroxybenzoate.
 6. A compound,(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamideor a pharmaceutically acceptable salt thereof containing less than 25%(2R,3R)-, (2S,3S)-, or(2R,3S)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamide,either individually or in combination, by weight.
 7. The compound ofclaim 6, containing less than 15% (2R,3R)-, (2S,3S)-, or(2R,3S)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamide,either individually or in combination, by weight.
 8. The compound ofclaim 6, containing less than 5% (2R,3R)-, (2S,3S)-, or(2R,3S)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamide,either individually or in combination, by weight.
 9. The compound ofclaim 6, containing less than 2% (2R,3R)-, (2S,3S)-, or(2R,3S)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamide,either individually or in combination, by weight.
 10. The compound ofclaim 6, containing less than 1% (2R,3R)-, (2S,3S)-, or(2R,3S)-N-2-((3-pyridinyl)methyl-1-azabicyclo[2.2.2]oct-3-yl)-3,5-difluorobenzamide,either individually or in combination, by weight.
 11. A compound(2S,3R)-N-2-((3-pyridinyl)methyl-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamide(Formula I) or a pharmaceutically acceptable salt thereof

which is substantially crystalline.
 12. A polymorphic form of a compound(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamidehydrochloride characterized by an x-ray diffraction pattern comprisingone or more peaks within ±0.5 degrees 2θ of the following peaks: 2θ 8.48.8 11.9 13.2 15.2 16.0 17.6 18.4 18.9 19.9 20.1 21.3 23.1 25.4 26.2.


13. A polymorphic form of a compound(2S,3R)-N-2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl-3,5-difluorobenzamidehydrochloride characterized by an x-ray powder diffraction pattern thatsubstantially corresponds to FIG.
 9. 14. A method for treating a diseaseor dysfunction, comprising administering a therapeutically effectiveamount of a compound of claim 1, wherein the disease or dysfunction isearly onset Alzheimer's disease, dementia of the Alzheimer's type, mildto moderate dementia of the Alzheimer's type, Alzheimer's disease,dyslexia, schizophrenia, schizophreniform disorder, schizoaffectivedisorder, cognitive deficits in schizophrenia, and cognitive dysfunctionin schizophrenia.
 15. A pharmaceutical composition comprising a compoundof claim 1, and one or more pharmaceutically acceptable carrier.